Modulation of Ephrin-B2 expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of Ephrin-B2. The compositions comprise oligonucleotides, targeted to nucleic acid encoding Ephrin-B2. Methods of using these compounds for modulation of Ephrin-B2 expression and for diagnosis and treatment of disease associated with expression of Ephrin-B2 are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods for modulating the expression of Ephrin-B2. In particular, this invention relates to compounds, particularly oligonucleotide compounds, which, in preferred embodiments, hybridize with nucleic acid molecules encoding Ephrin-B2. Such compounds are shown herein to modulate the expression of Ephrin-B2.

BACKGROUND OF THE INVENTION

[0002] The Eph and Eph-related receptors comprise the largest subfamily of receptor protein-tyrosine kinases and together with their ligands, the ephrins, they are implicated in mediating developmental events, particularly in the nervous system. The Eph receptors are named for their expression in an erythtopoietin-producing human hepatocellular carcinoma cell line while the ephrins are an abbreviation of Eph family receptor interacting proteins. Based on structures and sequence relationships, ephrins are divided into the ephrin-A (EFNA) class, which are anchored to the membrane by a glycosylphosphatidylinositol linkage, and the ephrin-B (EFNB) class, which are transmembrane proteins. The Eph family of receptors are divided into 2 groups, EphA and EphB, based on the similarity of their extracellular domain sequences and their preferential (but not exclusive) interaction with ephrin-A or ephrin-B ligands (Cheng et al., Cytokine Growth Factor Rev., 2002, 13, 75-85; Committee, Cell, 1997, 90, 403-404).

[0003] One of these ephrins, ephrin-B2, is expressed in developing arteries and is essential for embryonic heart development, and angiogenesis, as well as guiding axons in the developing nervous system (Gerety et al., Mol. Cell, 1999, 4, 403-414; Wang et al., Cell, 1998, 93, 741-753; Zhou et al., J. Neurosci. Res., 2001, 66, 1054-1063). The gene encoding ephrin-B2 (also called EFNB2, eph-related receptor tyrosine kinase ligand 5, EPLG5, ligand of eph-related kinase 5, LERK5, LERK-5, HTK ligand, HTKL, and HTK-L) was cloned simultaneously by two groups (Bennett et al., Proc. Natl. Acad. Sci. U.S. A., 1995, 92, 1866-1870; Cerretti et al., Mol. Immunol., 1995, 32, 1197-1205). Disclosed and claimed in U.S. Pat. No. 6,303,769 is an isolated DNA that encodes an ephrin-B2 protein, as well as an expression vector comprising said DNA, and a host cell transformed with said vector (Cerretti and Reddy, 2001).

[0004] One of the functions of Eph receptors and ephrins is to limit cell intermingling (Mellitzer et al., Nature, 1999, 400, 77-81), a function which is essential for the appropriate formation of veins and arteries, as well as the formation of synapses in the nervous system. The EphB4/ephrin-B2 signaling pathway has been recognized as a novel regulatory system for erythropoiesis where EphB4 is expressed on bone marrow erythroid progenitors and ephrin-B2 is expressed in bone marrow stromal cells (Inada et al., Blood, 1997, 89, 2757-2765). This complementary interaction is also seen in angiogenesis, where ephrin-B2, specifically expressed in the arterial endothelium, and EphB4, expressed in the venous endothelium, mediate reciprocal interactions between arterial and venous endothelial cells to aid in the formation of veins and arteries (zhang et al., Blood, 2001, 98, 1028-1037). Ephrin-B2 may have a further role in the formation of the arterial muscle wall as the expression in adults extends from the arterial endothelium into the surrounding smooth muscle cells (Gale et al., Dev. Biol., 2001, 230, 151-160). The expression of ephrin-B2 in epithelial, endothelial, and mesangial cells, accompanied by the expression of EphB4 in venous structures, may also serve to establish the glomerular microvasculature assembly (Takahashi et al., J. Am. Soc. Nephrol., 2001, 12, 2673-2682). This participation in angiogenesis may extend to inflammatory angiogenesis seen in periodontitis, where ephrin-B2 expression is upregulated (Yuan et al., J. Periodontal Res., 2000, 35, 165-171).

[0005] The interaction of ephrin-B2 with several Eph receptors is important in several brain functions, including proper development, cellular proliferation, and neuron signaling. The complementary expression of EphA4 and EphB1 receptors and ephrin-B2 is involved in restricting the intermingling of branchial crest neurons so that these neurons migrate to the appropriate locations for developing skeletal structures (Smith et al., Curr. Biol., 1997, 7, 561-570). Signaling mediated by ephrin-B2 with the Eph receptors EphB1, EphB2, and EphB3 has been shown to effect the migration of neuroblasts in the subventricular zone and may be a mediator of cell proliferation in the adult brain (Conover et al., Nat. Neurosci., 2000, 3, 1091-1097). Ephrin-B2 can also modulate NMDA receptor activity, a neurotransmitter receptor which, as a calcium ion channel, can initiate a biochemical response that results in modulation of synaptic strength (Takasu et al., Science, 2002, 295, 491-495). The ephrin-B2/EphB1 interaction may play a role in drug-induced plasticity in adults, as ephrin-B2 expression is upregulated by cocaine and amphetamines (Yue et al., J. Neurosci., 1999, 19, 2090-2101).

[0006] The normal activity of ephrins and Eph receptors in mediating cell-contact-dependent interactions is essential for normal development, however, aberrant activity has been implicated in several diseases such as cancer, possibly due to the ability of ephrin-B2 to promote angiogenesis. Ephrin-B2 and EphB4 have been examined in the morphogenesis of the normal and malignant mammary gland and the deregulated expression may contribute to mammary carcinogenesis (Nikolova et al., J. Cell Sci., 1998, 111, 2741-2751). The expression of ephrin-B2 and the receptors EphB2, EphB3, and EphB4 is higher in colon carcinoma specimens than in adjacent normal mucosa (Liu et al., Cancer, 2002, 94, 934-939), and increased expression is also associated with endometrial cancer (Takai et al., Oncol. Rep., 2001, 8, 567-573), small cell lung carcinoma (Tang et al., Clin. Cancer Res., 1999, 5, 455-460), leukemia-lymphoma (Steube et al., Leuk Lymphoma, 1999, 33, 371-376), and malignant melanomas (Vogt et al., Clin. Cancer Res., 1998, 4, 791-797).

[0007] Currently, there are no known therapeutic agents which effectively inhibit the synthesis of ephrin-B2 and to date, investigative strategies aimed at modulating ephrin-B2 function have involved the use of inactive mutants and knockout mice.

[0008] In mice, replacement of the endogenous ephrin-B2 gene with a carboxy-truncated ephrin-B2, or an ephrin-B2 lacking the cytoplasmic domain was used to examine the distinct function of each domain in the developing vertebrate embryo (Adams et al., Cell, 2001, 104, 57-69). Ephrin-B2 knockout mice have been generated to study the role of ephrin-B2 in regulating synaptic function (Henderson et al., Neuron, 2001, 32, 1041-1056) and angiogenesis (Wang et al., Cell, 1998, 93, 741-753).

[0009] Consequently, there remains a long felt need for additional agents capable of effectively inhibiting ephrin-B2 function.

[0010] Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of ephrin-B2 expression.

[0011] The present invention provides compositions and methods for modulating ephrin-B2 expression.

SUMMARY OF THE INVENTION

[0012] The present invention is directed to compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding Ephrin-B2, and which modulate the expression of Ephrin-B2. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of screening for modulators of Ephrin-B2 and methods of modulating the expression of Ephrin-B2 in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more of the compounds or compositions of the invention. Methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of Ephrin-B2 are also set forth herein. Such methods comprise administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the person in need of treatment.

DETAILED DESCRIPTION OF THE INVENTION

[0013] A. Overview of the Invention

[0014] The present invention employs compounds, preferably oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding Ephrin-B2. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding Ephrin-B2. As used herein, the terms “target nucleic acid” and “nucleic acid molecule encoding Ephrin-B2” have been used for convenience to encompass DNA encoding Ephrin-B2, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. The hybridization of a compound of this invention with its target nucleic acid is generally referred to as “antisense”. Consequently, the preferred mechanism believed to be included in the practice of some preferred embodiments of the invention is referred to herein as “antisense inhibition.” Such antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.

[0015] The functions of DNA to be interfered with can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. One preferred result of such interference with target nucleic acid function is modulation of the expression of Ephrin-B2. In the context of the present invention, “modulation” and “modulation of expression” mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition is often the preferred form of modulation of expression and mRNA is often a preferred target nucleic acid.

[0016] In the context of this invention, “hybridization” means the pairing of complementary strands of oligomeric compounds. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.

[0017] An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.

[0018] In the present invention the phrase “stringent hybridization conditions” or “stringent conditions” refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances and in the context of this invention, “stringent conditions” under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated.

[0019] “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleobases of an oligomeric compound. For example, if a nucleobase at a certain position of an oligonucleotide (an oligomeric compound), is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid.

[0020] It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). It is preferred that the antisense compounds of the present invention comprise at least 70% sequence complementarity to a target region within the target nucleic acid, more preferably that they comprise 90% sequence complementarity and even more preferably comprise 95% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0021] B. Compounds of the Invention

[0022] According to the present invention, compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid. One non-limiting example of such an enzyme is RNAse H, a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.

[0023] While the preferred form of antisense compound is a single-stranded antisense oligonucleotide, in many species the introduction of double-stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals and is believed to have an evolutionary connection to viral defense and transposon silencing.

[0024] The first evidence that dsRNA could lead to gene silencing in animals came in 1995 from work in the nematode, Caenorhabditis elegans (Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et al. have shown that the primary interference effects of dsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanism defined in Caenorhabditis elegans resulting from exposure to double-stranded RNA (dsRNA) has since been designated RNA interference (RNAi). This term has been generalized to mean antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of endogenous targeted mRNA levels (Fire et al., Nature, 1998, 391, 806-811). Recently, it has been shown that it is, in fact, the single-stranded RNA oligomers of antisense polarity of the dsRNAs which are the potent inducers of RNAi (Tijsterman et al., Science, 2002, 295, 694-697).

[0025] In the context of this invention, the term “oligomeric compound” refers to a polymer or oligomer comprising a plurality of monomeric units. In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.

[0026] While oligonucleotides are a preferred form of the compounds of this invention, the present invention comprehends other families of compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those described herein.

[0027] The compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). One of ordinary skill in the art will appreciate that the invention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.

[0028] In one preferred embodiment, the compounds of the invention are 12 to 50 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases in length.

[0029] In another preferred embodiment, the compounds of the invention are 15 to 30 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

[0030] Particularly preferred compounds are oligonucleotides from about 12 to about 50 nucleobases, even more preferably those comprising from about 15 to about 30 nucleobases.

[0031] Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.

[0032] Exemplary preferred antisense compounds include oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately upstream of the 5′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). Similarly preferred antisense compounds are represented by oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately downstream of the 3′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds.

[0033] C. Targets of the Invention

[0034] “Targeting” an antisense compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose function is to be modulated. This target nucleic acid may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target nucleic acid encodes Ephrin-B2.

[0035] The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., modulation of expression, will result. Within the context of the present invention, the term “region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Within regions of target nucleic acids are segments. “Segments” are defined as smaller or sub-portions of regions within a target nucleic acid. “Sites,” as used in the present invention, are defined as positions within a target nucleic acid.

[0036] Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding Ephrin-B2, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

[0037] The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon. Consequently, the “start codon region” (or “translation initiation codon region”) and the “stop codon region” (or “translation termination codon region”) are all regions which may be targeted effectively with the antisense compounds of the present invention.

[0038] The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, a preferred region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.

[0039] Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene), and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA (or corresponding nucleotides on the gene). The 5′ cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap site. It is also preferred to target the 5′ cap region.

[0040] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, may also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred target sites. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.

[0041] It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants”. More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence.

[0042] Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller “mRNA variants”. Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants”. If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

[0043] It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Within the context of the invention, the types of variants described herein are also preferred target nucleic acids.

[0044] The locations on the target nucleic acid to which the preferred antisense compounds hybridize are hereinbelow referred to as “preferred target segments.” As used herein the term “preferred target segment” is defined as at least an 8-nucleobase portion of a target region to which an active antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target segments represent portions of the target nucleic acid which are accessible for hybridization.

[0045] While the specific sequences of certain preferred target segments are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred target segments may be identified by one having ordinary skill.

[0046] Target segments 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target segments are considered to be suitable for targeting as well.

[0047] Target segments can include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly preferred target segments are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred target segments illustrated herein will be able, without undue experimentation, to identify further preferred target segments.

[0048] Once one or more target regions, segments or sites have been identified, antisense compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

[0049] D. Screening and Target Validation

[0050] In a further embodiment, the “preferred target segments” identified herein may be employed in a screen for additional compounds that modulate the expression of Ephrin-B2. “Modulators” are those compounds that decrease or increase the expression of a nucleic acid molecule encoding Ephrin-B2 and which comprise at least an 8-nucleobase portion which is complementary to a preferred target segment. The screening method comprises the steps of contacting a preferred target segment of a nucleic acid molecule encoding Ephrin-B2 with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding Ephrin-B2. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the expression of a nucleic acid molecule encoding Ephrin-B2, the modulator may then be employed in further investigative studies of the function of Ephrin-B2, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.

[0051] The preferred target segments of the present invention may be also be combined with their respective complementary antisense compounds of the present invention to form stabilized double-stranded (duplexed) oligonucleotides.

[0052] Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processsing via an antisense mechanism. Moreover, the double-stranded moieties may be subject to chemical modifications (Fire et al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200). For example, such double-stranded moieties have been shown to inhibit the target by the classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target (Tijsterman et al., Science, 2002, 295, 694-697).

[0053] The compounds of the present invention can also be applied in the areas of drug discovery and target validation. The present invention comprehends the use of the compounds and preferred target segments identified herein in drug discovery efforts to elucidate relationships that exist between Ephrin-B2 and a disease state, phenotype, or condition. These methods include detecting or modulating Ephrin-B2 comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of Ephrin-B2 and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound of the invention. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotype.

[0054] E. Kits, Research Reagents, Diagnostics, and Therapeutics

[0055] The compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. Furthermore, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.

[0056] For use in kits and diagnostics, the compounds of the present invention, either alone or in combination with other compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.

[0057] As one nonlimiting example, expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.

[0058] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S. A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SURF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0059] The compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding Ephrin-B2. For example, oligonucleotides that are shown to hybridize with such efficiency and under such conditions as disclosed herein as to be effective Ephrin-B2 inhibitors will also be effective primers or probes under conditions favoring gene amplification or detection, respectively. These primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding Ephrin-B2 and in the amplification of said nucleic acid molecules for detection or for use in further studies of Ephrin-B2. Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding Ephrin-B2 can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of Ephrin-B2 in a sample may also be prepared.

[0060] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that antisense compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.

[0061] For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of Ephrin-B2 is treated by administering antisense compounds in accordance with this invention. For example, in one non-limiting embodiment, the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of a Ephrin-B2 inhibitor. The Ephrin-B2 inhibitors of the present invention effectively inhibit the activity of the Ephrin-B2 protein or inhibit the expression of the Ephrin-B2 protein. In one embodiment, the activity or expression of Ephrin-B2 in an animal is inhibited by about 10%. Preferably, the activity or expression of Ephrin-B2 in an animal is inhibited by about 30%. More preferably, the activity or expression of Ephrin-B2 in an animal is inhibited by 50% or more.

[0062] For example, the reduction of the expression of Ephrin-B2 may be measured in serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal. Preferably, the cells contained within said fluids, tissues or organs being analyzed contain a nucleic acid molecule encoding Ephrin-B2 protein and/or the Ephrin-B2 protein itself.

[0063] The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.

[0064] F. Modifications

[0065] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

[0066] Modified Internucleoside Linkages (Backbones)

[0067] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0068] Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphoro-dithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 51 or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 31 linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0069] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0070] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0071] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0072] Modified Sugar and Internucleoside Linkages-Mimetics

[0073] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage (i.e. the backbone), of the nucleotide units are replaced with novel groups. The nucleobase units are maintained for hybridization with an appropriate target nucleic acid. One such compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0074] Preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0075] Modified Sugars

[0076] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂) CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂) CH₃]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF3, OCF₃, SOCH₃. SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂) 20N(CH₃)₂ group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

[0077] Other preferred modifications include 21-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0078] A further preferred modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. The linkage is preferably a methylene (—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

[0079] Natural and Modified Nucleobases

[0080] Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

[0081] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

[0082] Conjugates

[0083] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, the entire disclosure of which are incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed June 15, 1999) which is incorporated herein by reference in its entirety.

[0084] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

[0085] Chimeric Compounds

[0086] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.

[0087] The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as RNAseL which cleaves both cellular and viral RNA. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0088] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0089] G. Formulations

[0090] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

[0091] The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0092] The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0093] The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. For oligonucleotides, preferred examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0094] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

[0095] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0096] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0097] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.

[0098] Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0099] Formulations of the present invention include liposomal formulations. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.

[0100] Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0101] The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0102] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0103] One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.

[0104] Preferred formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).

[0105] For topical or other administration, oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999, which is incorporated herein by reference in its entirety.

[0106] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts and fatty acids and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. Nos. 09/108,673 (filed Jul. 1, 1998), 09/315,298 (filed May 20, 1999) and 10/071,822, filed Feb. 8, 2002, each of which is incorporated herein by reference in their entirety.

[0107] Compositions and formulations for parenteral, intra-thecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0108] Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligomeric compounds and one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of antisense compounds and other non-antisense drugs are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0109] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Alternatively, compositions of the invention may contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

[0110] H. Dosing

[0111] The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0112] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

EXAMPLES Example 1

[0113] Synthesis of Nucleoside Phosphoramidites

[0114] The following compounds, including amidites and their intermediates were prepared as described in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-N-4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N-4-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine, 2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modified amidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate, 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite), 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine intermediate, 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidine penultimate intermediate, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylamino-oxyethyl) nucleoside amidites, 2′-(Dimethylaminooxyethoxy) nucleoside amidites, 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine, 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine, 2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-(Aminooxyethoxy) nucleoside amidites, N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites, 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine, 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine and 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2

[0115] Oligonucleotide and Oligonucleoside Synthesis

[0116] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0117] Oligonucleotides: Unsubstituted and Substituted

[0118] phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.

[0119] Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

[0120] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

[0121] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.

[0122] Phosphoramidite oligonucleotides are prepared as described in U.S. Patent, 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

[0123] Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

[0124] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0125] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

[0126] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

[0127] Oligonucleosides: Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligo-nucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

[0128] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

[0129] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 3

[0130] RNA Synthesis

[0131] In general, RNA synthesis chemistry is based on the selective incorporation of various protecting groups at strategic intermediary reactions. Although one of ordinary skill in the art will understand the use of protecting groups in organic synthesis, a useful class of protecting groups includes silyl ethers. In particular bulky silyl ethers are used to protect the 5′-hydroxyl in combination with an acid-labile orthoester protecting group on the 2′-hydroxyl. This set of protecting groups is then used with standard solid-phase synthesis technology. It is important to lastly remove the acid labile orthoester protecting group after all other synthetic steps. Moreover, the early use of the silyl protecting groups during synthesis ensures facile removal when desired, without undesired deprotection of 2′ hydroxyl.

[0132] Following this procedure for the sequential protection of the 5′-hydroxyl in combination with protection of the 2′-hydroxyl by protecting groups that are differentially removed and are differentially chemically labile, RNA oligonucleotides were synthesized.

[0133] RNA oligonucleotides are synthesized in a stepwise fashion. Each nucleotide is added sequentially (3′- to 5′-direction) to a solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are added, coupling the second base onto the 5′-end of the first nucleoside. The support is washed and any unreacted 5′-hydroxyl groups are capped with acetic anhydride to yield 5′-acetyl moieties. The linkage is then oxidized to the more stable and ultimately desired P(V) linkage. At the end of the nucleotide addition cycle, the 5′-silyl group is cleaved with fluoride. The cycle is repeated for each subsequent nucleotide.

[0134] Following synthesis, the methyl protecting groups on the phosphates are cleaved in 30 minutes utilizing 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂) in DMF. The deprotection solution is washed from the solid support-bound oligonucleotide using water. The support is then treated with 40% methylamine in water for 10 minutes at 55° C. This releases the RNA oligonucleotides into solution, deprotects the exocyclic amines, and modifies the 2′-groups. The oligonucleotides can be analyzed by anion exchange HPLC at this stage.

[0135] The 2′-orthoester groups are the last protecting groups to be removed. The ethylene glycol monoacetate orthoester protecting group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is one example of a useful orthoester protecting group which, has the following important properties. It is stable to the conditions of nucleoside phosphoramidite synthesis and oligonucleotide synthesis. However, after oligonucleotide synthesis the oligonucleotide is treated with methylamine which not only cleaves the oligonucleotide from the solid support but also removes the acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxyl substituents on the orthoester are less electron withdrawing than the acetylated precursor. As a result, the modified orthoester becomes more labile to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is approximately 10 times faster after the acetyl groups are removed. Therefore, this orthoester possesses sufficient stability in order to be compatible with oligonucleotide synthesis and yet, when subsequently modified, permits deprotection to be carried out under relatively mild aqueous conditions compatible with the final RNA oligonucleotide product.

[0136] Additionally, methods of RNA synthesis are well known in the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331).

[0137] RNA antisense compounds (RNA oligonucleotides) of the present invention can be synthesized by the methods herein or purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once synthesized, complementary RNA antisense compounds can then be annealed by methods known in the art to form double stranded (duplexed) antisense compounds. For example, duplexes can be formed by combining 30 μl of each of the complementary strands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and 15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90° C., then 1 hour at 37° C. The resulting duplexed antisense compounds can be used in kits, assays, screens, or other methods to investigate the role of a target nucleic acid.

Example 4

[0138] Synthesis of Chimeric Oligonucleotides

[0139] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.

[0140] [2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric Phosphorothioate oligonucleotides

[0141] Chimeric oligonucleotides having 21-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligo-nucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 51-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spetrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

[0142] [2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides

[0143] [2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites.

[0144] [2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxy Phosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides

[0145] [2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxy phosphorothioate]-[2′-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

[0146] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5

[0147] Design and Screening of Duplexed Antisense Compounds Targeting Ephrin-B2

[0148] In accordance with the present invention, a series of nucleic acid duplexes comprising the antisense compounds of the present invention and their complements can be designed to target Ephrin-B2. The nucleobase sequence of the antisense strand of the duplex comprises at least a portion of an oligonucleotide in Table 1. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the dsRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini.

[0149] For example, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG and having a two-nucleobase overhang of deoxythymidine(dT) would have the following structure:   cgagaggcggacgggaccgTT Antisense Strand   ||||||||||||||||||| TTgctctccgcctgccctggc Complement

[0150] RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.). Once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 μM. Once diluted, 30 uL of each strand is combined with 15 uL of a 5× solution of annealing buffer. The final concentration of said buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 μM magnesium acetate. The final volume is 75 uL. This solution is incubated for 1 minute at 90° C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37° C. at which time the dsRNA duplexes are used in experimentation. The final concentration of the dsRNA duplex is 20 uM. This solution can be stored frozen (−20° C.) and freeze-thawed up to 5 times.

[0151] Once prepared, the duplexed antisense compounds are evaluated for their ability to modulate Ephrin-B2 expression.

[0152] When cells reached 80% confluency, they are treated with duplexed antisense compounds of the invention. For cells grown in 96-well plates, wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mL LIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at a final concentration of 200 nM. After 5 hours of treatment, the medium is replaced with fresh medium. Cells are harvested 16 hours after treatment, at which time RNA is isolated and target reduction measured by RT-PCR.

Example 6

[0153] Oligonucleotide Isolation

[0154] After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH₄OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the −16 amu product (+/−32+/−48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7

[0155] Oligonucleotide Synthesis—96 Well Plate Format

[0156] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

[0157] Oligonucleotides were cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8

[0158] Oligonucleotide Analysis—96-Well Plate Format

[0159] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.

Example 9

[0160] Cell Culture and Oligonucleotide Treatment

[0161] The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR.

[0162] T-24 Cells:

[0163] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #353872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0164] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

[0165] A549 Cells:

[0166] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

[0167] NHDF Cells:

[0168] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

[0169] HEK Cells:

[0170] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

[0171] Treatment with Antisense Compounds:

[0172] When cells reached 65-75% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEM™-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 μL of OPTI-EM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation, Carlsbad, Calif.) and the desired concentration of oligonucleotide. Cells are treated and data are obtained in triplicate. After 4-7 hours of treatment at 37° C., the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0173] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is selected from either ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are 2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. The concentrations of antisense oligonucleotides used herein are from 50 nM to 300 iNM.

Example 10

[0174] Analysis of Oligonucleotide Inhibition of Ephrin-B2 Expression

[0175] Antisense modulation of Ephrin-B2 expression can be assayed in a variety of ways known in the art. For example, Ephrin-B2 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0176] Protein levels of Ephrin-B2 can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS). Antibodies directed to Ephrin-B2 can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.

Example 11

[0177] Design of Phenotypic Assays and In Vivo Studies for the Use of Ephrin-B2 Inhibitors

[0178] Phenotypic Assays

[0179] Once Ephrin-B2 inhibitors have been identified by the methods disclosed herein, the compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition. Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of Ephrin-B2 in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).

[0180] In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with Ephrin-B2 inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.

[0181] Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.

[0182] Analysis of the geneotype of the cell (measurement of the expression of one or more of the genes of the cell) after treatment is also used as an indicator of the efficacy or potency of the Ephrin-B2 inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.

[0183] In Vivo Studies

[0184] The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.

[0185] The clinical trial is subjected to rigorous controls to ensure that individuals are not unnecessarily put at risk and that they are fully informed about their role in the study. To account for the psychological effects of receiving treatments, volunteers are randomly given placebo or Ephrin-B2 inhibitor. Furthermore, to prevent the doctors from being biased in treatments, they are not informed as to whether the medication they are administering is a Ephrin-B2 inhibitor or a placebo. Using this randomization approach, each volunteer has the same chance of being given either the new treatment or the placebo.

[0186] Volunteers receive either the Ephrin-B2 inhibitor or placebo for eight week period with biological parameters associated with the indicated disease state or condition being measured at the beginning (baseline measurements before any treatment), end (after the final treatment), and at regular intervals during the study period. Such measurements include the levels of nucleic acid molecules encoding Ephrin-B2 or Ephrin-B2 protein levels in body fluids, tissues or organs compared to pre-treatment levels. Other measurements include, but are not limited to, indices of the disease state or condition being treated, body weight, blood pressure, serum titers of pharmacologic indicators of disease or toxicity as well as ADME (absorption, distribution, metabolism and excretion) measurements.

[0187] Information recorded for each patient includes age (years), gender, height (cm), family history of disease state or condition (yes/no), motivation rating (some/moderate/great) and number and type of previous treatment regimens for the indicated disease or condition.

[0188] Volunteers taking part in this study are healthy adults (age 18 to 65 years) and roughly an equal number of males and females participate in the study. Volunteers with certain characteristics are equally distributed for placebo and Ephrin-B2 inhibitor treatment. In general, the volunteers treated with placebo have little or no response to treatment, whereas the volunteers treated with the Ephrin-B2 inhibitor show positive trends in their disease state or condition index at the conclusion of the study.

Example 12

[0189] RNA Isolation

[0190] Poly(A)+ mRNA Isolation

[0191] Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine in the art. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0192] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

[0193] Total RNA Isolation

[0194] Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 140 μL of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.

[0195] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Example 13

[0196] Real-Time Quantitative PCR Analysis of Ephrin-B2 mRNA Levels

[0197] Quantitation of Ephrin-B2 mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0198] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.

[0199] PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of DATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0200] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

[0201] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.

[0202] Probes and primers to human Ephrin-B2 were designed to hybridize to a human Ephrin-B2 sequence, using published sequence information (GenBank accession number NM_(—004093.1), incorporated herein as SEQ ID NO:4). For human Ephrin-B2 the PCR primers were:

[0203] forward primer: CTTGGAGGGAAGGGAGAAAGTAG (SEQ ID NO: 5)

[0204] reverse primer: TGTGTATCTTATTGCCAGTACCACAA (SEQ ID NO: 6) and

[0205] the PCR probe was: FAM-CCGCTGATGATATATTCGGGCAGGACT-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were:

[0206] forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8)

[0207] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the

[0208] PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC— TAMRA 3′ (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14

[0209] Northern Blot Analysis of Ephrin-B2 mRNA Levels

[0210] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0211] To detect human Ephrin-B2, a human Ephrin-B2 specific probe was prepared by PCR using the forward primer CTTGGAGGGAAGGGAGAAAGTAG (SEQ ID NO: 5) and the reverse primer TGTGTATCTTATTGCCAGTACCACAA (SEQ ID NO: 6). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0212] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15

[0213] Antisense Inhibition of Human Ephrin-B2 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

[0214] In accordance with the present invention, a series of antisense compounds were designed to target different regions of the human Ephrin-B2 RNA, using published sequences (GenBank accession number NM_(—)004093.1, incorporated herein as SEQ ID NO: 4, the complement of nucleotides 54000 to 103000 of the sequence with GenBank accession number NT_(—)009891.5, incorporated herein as SEQ ID NO: 11, and GenBank accession number AA308967.1, incorporated herein as SEQ ID NO: 12). The compounds are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the compound binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human Ephrin-B2 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments in which T-24 cells were treated with the antisense oligonucleotides of the present invention. If present, “N.D.” indicates “no data”. TABLE 1 Inhibition of human Ephrin-B2 mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TARGET SEQ ID TARGET SEQ ID ISIS # REGION NO SITE SEQUENCE % INHIB NO 155134 3′UTR 4 1318 aaccactgtcttcccttggc 49 13 155135 3′UTR 4 2331 aagtgtcgtaaggctcaatc 61 14 155136 3′UTR 4 2248 ttaataacaacgatgatgat 0 15 155137 3′UTR 4 2098 tcatccaaagcagaccgact 0 16 155138 Coding 4 199 caacagttttagagtccact 68 17 155139 3′UTR 4 2047 ccagtaccacaacagtcctg 88 18 155140 3′UTR 4 2035 cagtcctgcccgaatatatc 0 19 155141 3′UTR 4 1874 ccactgtgtgtgctgtcgtg 93 20 155142 3′UTR 4 2327 gtcgtaaggctcaatcggga 52 21 155143 3′UTR 4 2801 atgatgcagtcacatcggct 77 22 155144 Coding 4 712 tgatgacgatgaagatgatg 38 23 155145 3′UTR 4 1351 cctgccaggatgctcacagc 70 24 155146 3′UTR 4 1664 taaagggcaccttgttagca 83 25 155147 Coding 4 129 tccttgtccaggtagaaatt 0 26 155148 3′UTR 4 2727 gaggacttggttatttttgc 80 27 155149 Coding 4 469 ggatcttcatggctcttgtc 41 28 155150 3′UTR 4 2393 gctgcaggctccatcagtac 51 29 155151 3′UTR 4 1575 ccagacatgagtgttccatg 93 30 155152 3′UTR 4 1045 ggcatcgggacattaggtgt 49 31 155153 3′UTR 4 2153 tttccctgtgtttaataaaa 48 32 155154 3′UTR 4 1776 ctttctgtttcagaggcagc 82 33 155155 Coding 4 499 cagaacttgcatcttgtcca 40 34 155156 3′UTR 4 2883 tctagagaacacttcagccc 42 35 155157 3′UTR 4 2507 ctattataaatacgtcctat 15 36 155158 3′UTR 4 1060 caaaccctcaagggaggcat 53 37 155159 Coding 4 12 cacggagtcccttctcacag 60 38 155160 Stop 4 1001 cagggtccctctcagacctt 0 39 Codon 155161 3′UTR 4 1033 ttaggtgtcctctgggaaag 52 40 155162 3′UTR 4 1336 acagccctctcgtccacaaa 10 41 155163 3′UTR 4 1314 actgtcttcccttggcttct 44 42 155164 Coding 4 362 tctagaccccagaggttagg 76 43 155165 Coding 4 308 tcttggtctggtttggcaca 72 44 155166 Coding 4 116 agaaatttggagttcgagga 0 45 155167 3′UTR 4 2087 agaccgactctcctacagct 52 46 155168 Coding 4 684 agcaatccctgcaaataagg 0 47 155169 3′UTR 4 1216 tctgcacagtcttccagctt 31 48 155170 Coding 4 120 aggtagaaatttggagttcg 0 49 216481 Start 4 1 ttctcacagccatggctgtg 14 50 Codon 216482 Coding 4 66 tttggaaatcgcagttctgc 82 51 216483 Coding 4 144 tgggtatagtaccagtcct 94 52 216484 Coding 4 325 tgatggtgaatttgatatct 62 53 216485 Coding 4 406 catttgatgtagatataatg 30 54 216486 Coding 4 610 tgctagaacctggatttggt 77 55 216487 3′UTR 4 1493 gagtgagacagtaaggacta 74 56 216488 3′UTR 4 1524 tgaggtgctgcagagccctg 90 57 216489 3′UTR 4 1630 gaacggattacccatggact 89 58 216490 3′UTR 4 2363 gccctggcacccggacagca 77 59 216491 3′UTR 4 2471 gacatcataatttacttccg 85 60 216492 3′UTR 4 2864 cataaggcaagggaaaaccc 69 61 216493 intron 11 28364 gatgtgtcttagctatggat 25 62 216494 intron 11 32504 cactggactctctctttcct 54 63 216495 intron 11 34358 gctaggattacaggcgtgag 40 64 216496 intron 11 35882 tggcacagagatgtgaaacg 75 65 216497 exon: 11 40477 tggtctttaccttgtccaac 15 66 intron junction 216498 intron: 11 41230 gaacttgcatctatatgaaa 0 67 exon junction 216499 exon: 11 41344 gctgttatacctggatttgg 23 68 intron junction 216500 intron: 11 42796 gtgctagaacctgcagacgc 46 69 exon junction 216501 exon 11 45092 agcccccagtgtgacctgct 91 70 216502 exon 11 45117 gagccacagctaaatcgtca 78 71 216503 exon 11 45369 tttccaggttcaggaaatag 62 72 216504 exon 11 45455 attgtagatatatattatac 0 73 216505 exon 11 45519 tgtactgtggctggttacca 55 74 216506 exon 11 45525 tacatatgtactgtggctgg 56 75 216507 exon 11 45575 aactcttgcatgaatgcaca 88 76 216508 exon 11 45612 cccccatcctaaaagtaact 0 77 216509 exon 11 45949 taaaatctgaacctatttac 31 78 216510 exon 11 46061 attgctctatgtacacaatc 10 79 216511 exon 11 46198 ggcaaccttttataccattt 47 80 216512 exon 11 46217 cacaaatatgcagcaatttg 50 81 216513 exon 11 46355 agcagatttaaaacctatga 43 82 216514 exon 11 46371 tgcaatgtgaaactaaagca 63 83 216515 genomic 12 64 cgcaggctgggacccccaat 87 84

[0215] As shown in Table 1, SEQ ID NOs 13, 14, 17, 18, 20, 21, 22, 24, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 37, 38, 40, 42, 43, 44, 46, 51, 52, 53, 55, 56, 57, 58, 59, 60, 61, 63, 64, 65, 69, 70, 71, 72, 74, 75, 76, 80, 81, 82, 83 and 84 demonstrated at least 40% inhibition of human Ephrin-B2 expression in this assay and are therefore preferred. More preferred are SEQ ID NOs 25, 20 and 30. The target regions to which these preferred sequences are complementary are herein referred to as “preferred target segments” and are therefore preferred for targeting by compounds of the present invention. These preferred target segments are shown in Table 2. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 2 is the species in which each of the preferred target segments was found. TABLE 2 Sequence and position of preferred target segments identified in Ephrin-B2. TARGET SITE SEQ ID TARGET REV COMP SEQ ID ID NO SITE SEQUENCE OF SEQ ID ACTIVE IN NO 70635 4 1318 gccaagggaagacagtggtt 13 H. sapiens 85 70636 4 2331 gattgagccttacgacactt 14 H. sapiens 86 70639 4 199 agtggactctaaaactgttg 17 H. sapiens 87 70640 4 2047 caggactgttgtggtactgg 18 H. sapiens 88 70642 4 1874 cacgacagcacacacagtgg 20 H. sapiens 89 70643 4 2327 tcccgattgagccttacgac 21 H. sapiens 90 70644 4 2801 agccgatgtgactgcatcat 22 H. sapiens 91 70646 4 1351 gctgtgagcatcctggcagg 24 H. sapiens 92 70647 4 1664 tgctaacaaggtgcccttta 25 H. sapiens 93 70649 4 2727 gcaaaaataaccaagtcctc 27 H. sapiens 94 70650 4 469 gacaagagccatgaagatcc 28 H. sapiens 95 70651 4 2393 gtactgatggagectgcagc 29 H. sapiens 96 70652 4 1575 catggaacactcatgtctgg 30 H. sapiens 97 70653 4 1045 acacctaatgtcccgatgcc 31 H. sapiens 98 70654 4 2153 ttttattaaacacagggaaa 32 H. sapiens 99 70655 4 1776 gctgcctctgaaacaqaaag 33 H. sapiens 100 70656 4 499 tggacaagatgcaagttctg 34 H. sapiens 101 70657 4 2883 gggctgaagtgttctctaga 35 H. sapiens 102 70659 4 1060 atgcctcccttgagggtttg 37 H. sapiens 103 70660 4 12 ctgtgagaagggactccgtg 38 H. sapiens 104 70662 4 1033 ctttcccaqaggacacctaa 40 H. sapiens 105 70664 4 1314 agaagccaagggaagacagt 42 H. sapiens 106 70665 4 362 cctaacctctggggtctaga 43 H. sapiens 107 70666 4 308 tgtgccaaaccagaccaaga 44 H. sapiens 108 70668 4 2087 agctgtaggagagtcggtct 46 H. sapiens 109 133169 4 66 gcagaactgcgatttccaaa 51 H. sapiens 110 133170 4 144 aaggactggtactataccca 52 H. sapiens 111 133171 4 325 agatatcaaattcaccatca 53 H. sapiens 112 133173 4 610 accaaatccaggttctagca 55 H. sapiens 113 133174 4 1493 tagtccttactgtctcactc 56 H. sapiens 114 133175 4 1524 cagggctctgcagcacctca 57 H. sapiens 115 133176 4 1630 agtccatgggtaatccgttc 58 H. sapiens 116 133177 4 2363 tgctgtccgggtgccagggc 59 H. sapiens 117 133178 4 2471 cggaagtaaattatgatgtc 60 H. sapiens 118 133179 4 2864 gggttttcccttgccttatg 61 H. sapiens 119 133181 11 32504 aggaaagagagagtccagtg 63 H. sapiens 120 133182 11 34358 ctcacgcctgtaatcctagc 64 H. sapiens 121 133183 11 35882 cgtttcacatctctgtgcca 65 H. sapiens 122 133187 11 42796 gcgtctgcaggttctagcac 69 H. sapiens 123 133188 11 45092 agcaggtcacactgggggct 70 H. sapiens 124 133189 11 45117 tgacgatttagctgtggctc 71 H. sapiens 125 133190 11 45369 ctatttcctgaacctggaaa 72 H. sapiens 126 133192 11 45519 tggtaaccagccacagtaca 74 H. sapiens 127 133193 11 45525 ccagccacagtacatatgta 75 H. sapiens 128 133194 11 45575 tgtgcattcatgcaagagtt 76 H. sapiens 129 133198 11 46198 aaatggtataaaaqgttgcc 80 H. sapiens 130 133199 11 46217 caaattgctgcatatttgtg 81 H. sapiens 131 133200 11 46355 tcataggttttaaatctgct 82 H. sapiens 132 133201 11 46371 tgctttagtttcacattgca 83 H. sapiens 133 133202 12 64 attgggggtcccagcctgcg 84 H. sapiens 134

[0216] As these “preferred target segments” have been found by experimentation to be open to, and accessible for, hybridization with the antisense compounds of the present invention, one of skill in the art will recognize or be able to ascertain, using no more than routine experimentation, further embodiments of the invention that encompass other compounds that specifically hybridize to these preferred target segments and consequently inhibit the expression of Ephrin-B2.

[0217] According to the present invention, antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other short oligomeric compounds which hybridize to at least a portion of the target nucleic acid.

Example 16

[0218] Western Blot Analysis of Ephrin-B2 Protein Levels

[0219] Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to Ephrin-B2 is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

1 134 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1 tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence Antisense Oligonucleotide 2 gtgcgcgcga gcccgaaatc 20 3 20 DNA Artificial Sequence Antisense Oligonucleotide 3 atgcattctg cccccaagga 20 4 2902 DNA H. sapiens CDS (8)...(1009) 4 cacagcc atg gct gtg aga agg gac tcc gtg tgg aag tac tgc tgg ggt 49 Met Ala Val Arg Arg Asp Ser Val Trp Lys Tyr Cys Trp Gly 1 5 10 gtt ttg atg gtt tta tgc aga act gcg att tcc aaa tcg ata gtt tta 97 Val Leu Met Val Leu Cys Arg Thr Ala Ile Ser Lys Ser Ile Val Leu 15 20 25 30 gag cct atc tat tgg aat tcc tcg aac tcc aaa ttt cta cct gga caa 145 Glu Pro Ile Tyr Trp Asn Ser Ser Asn Ser Lys Phe Leu Pro Gly Gln 35 40 45 gga ctg gta cta tac cca cag ata gga gac aaa ttg gat att att tgc 193 Gly Leu Val Leu Tyr Pro Gln Ile Gly Asp Lys Leu Asp Ile Ile Cys 50 55 60 ccc aaa gtg gac tct aaa act gtt ggc cag tat gaa tat tat aaa gtt 241 Pro Lys Val Asp Ser Lys Thr Val Gly Gln Tyr Glu Tyr Tyr Lys Val 65 70 75 tat atg gtt gat aaa gac caa gca gac aga tgc act att aag aag gaa 289 Tyr Met Val Asp Lys Asp Gln Ala Asp Arg Cys Thr Ile Lys Lys Glu 80 85 90 aat acc cct ctc ctc aac tgt gcc aaa cca gac caa gat atc aaa ttc 337 Asn Thr Pro Leu Leu Asn Cys Ala Lys Pro Asp Gln Asp Ile Lys Phe 95 100 105 110 acc atc aag ttt caa gaa ttc agc cct aac ctc tgg ggt cta gaa ttt 385 Thr Ile Lys Phe Gln Glu Phe Ser Pro Asn Leu Trp Gly Leu Glu Phe 115 120 125 cag aag aac aaa gat tat tac att ata tct aca tca aat ggg tct ttg 433 Gln Lys Asn Lys Asp Tyr Tyr Ile Ile Ser Thr Ser Asn Gly Ser Leu 130 135 140 gag ggc ctg gat aac cag gag gga ggg gtg tgc cag aca aga gcc atg 481 Glu Gly Leu Asp Asn Gln Glu Gly Gly Val Cys Gln Thr Arg Ala Met 145 150 155 aag atc ctc atg aaa gtt gga caa gat gca agt tct gct gga tca acc 529 Lys Ile Leu Met Lys Val Gly Gln Asp Ala Ser Ser Ala Gly Ser Thr 160 165 170 agg aat aaa gat cca aca aga cgt cca gaa cta gaa gct ggt aca aat 577 Arg Asn Lys Asp Pro Thr Arg Arg Pro Glu Leu Glu Ala Gly Thr Asn 175 180 185 190 gga aga agt tcg aca aca agt ccc ttt gta aaa cca aat cca ggt tct 625 Gly Arg Ser Ser Thr Thr Ser Pro Phe Val Lys Pro Asn Pro Gly Ser 195 200 205 agc aca gac ggc aac agc gcc gga cat tcg ggg aac aac atc ctc ggt 673 Ser Thr Asp Gly Asn Ser Ala Gly His Ser Gly Asn Asn Ile Leu Gly 210 215 220 tcc gaa gtg gcc tta ttt gca ggg att gct tca gga tgc atc atc ttc 721 Ser Glu Val Ala Leu Phe Ala Gly Ile Ala Ser Gly Cys Ile Ile Phe 225 230 235 atc gtc atc atc atc acg ctg gtg gtc ctc ttg ctg aag tac cgg agg 769 Ile Val Ile Ile Ile Thr Leu Val Val Leu Leu Leu Lys Tyr Arg Arg 240 245 250 aga cac agg aag cac tcg ccg cag cac acg acc acg ctg tcg ctc agc 817 Arg His Arg Lys His Ser Pro Gln His Thr Thr Thr Leu Ser Leu Ser 255 260 265 270 aca ctg gcc aca ccc aag cgc agc ggc aac aac aac ggc tca gag ccc 865 Thr Leu Ala Thr Pro Lys Arg Ser Gly Asn Asn Asn Gly Ser Glu Pro 275 280 285 agt gac att atc atc ccg cta agg act gcg gac agc gtc ttc tgc cct 913 Ser Asp Ile Ile Ile Pro Leu Arg Thr Ala Asp Ser Val Phe Cys Pro 290 295 300 cac tac gag aag gtc agc ggg gac tac ggg cac ccg gtg tac atc gtc 961 His Tyr Glu Lys Val Ser Gly Asp Tyr Gly His Pro Val Tyr Ile Val 305 310 315 cag gag atg ccc ccg cag agc ccg gcg aac att tac tac aag gtc tga 1009 Gln Glu Met Pro Pro Gln Ser Pro Ala Asn Ile Tyr Tyr Lys Val * 320 325 330 gagggaccct ggtggtacct gtgctttccc agaggacacc taatgtcccg atgcctccct 1069 tgagggtttg agagcccgcg tgctggagaa ttgactgaag cacagcaccg ggggagaggg 1129 acactcctcc tcggaagagc ccgtcgcgct ggacagctta cctagtcttg tagcattcgg 1189 ccttggtgaa cacacacgct ccctggaagc tggaagactg tgcagaagac gcccattcgg 1249 actgctgtgc cgcgtcccac gtctcctcct cgaagccatg tgctgcggtc actcaggcct 1309 ctgcagaagc caagggaaga cagtggtttg tggacgagag ggctgtgagc atcctggcag 1369 gtgccccagg atgccacgcc tggaagggcc ggcttctgcc tggggtgcat ttcccccgca 1429 gtgcataccg gacttgtcac acggacctcg ggctagttaa ggtgtgcaaa gatctctaga 1489 gtttagtcct tactgtctca ctcgttctgt tacccagggc tctgcagcac ctcacctgag 1549 acctccactc cacatctgca tcactcatgg aacactcatg tctggagtcc cctcctccag 1609 ccgctggcaa caacagcttc agtccatggg taatccgttc atagaaattg tgtttgctaa 1669 caaggtgccc tttagccaga tgctaggctg tctgcgaaga aggctaggag ttcatagaag 1729 ggagtggggc tggggaaagg gctggctgca attgcagctc actgctgctg cctctgaaac 1789 agaaagttgg aaaggaaaaa agaaaaaagc aattaggtag cacagcactt tggttttgct 1849 gagatcgaag aggccagtag gagacacgac agcacacaca gtggattcca gtgcatgggg 1909 aggcactcgc tgttatcaaa tagcgatgtg caggaagaaa agcccctctt cattccgggg 1969 aacaaagacg ggtattgttg ggaaaggaac aggcttggag ggaagggaga aagtaggccg 2029 ctgatgatat attcgggcag gactgttgtg gtactggcaa taagatacac agctccgagc 2089 tgtaggagag tcggtctgct ttggatgatt ttttaagcag actcagctgc tatacttatc 2149 acattttatt aaacacaggg aaagcattta ggagaatagc agagagccaa atctgaccta 2209 aaagttgaaa agccaaaggt caaacaggct gtaattccat catcatcgtt gttattaaag 2269 aatccttatc tataaaaggt aggtcagatc cccctccccc caggttcctc cttcccctcc 2329 cgattgagcc ttacgacact ttggtttatg cggtgctgtc cgggtgccag ggctgcaggg 2389 tcggtactga tggagcctgc agcgcccggt gctctgtgtc aaggtgaagc acatacggca 2449 gacctcttag agtccttaag acggaagtaa attatgatgt ccagggggag aaggaagata 2509 ggacgtattt ataataggta tatagaacac aagggatata aaatgaaaga tttttactaa 2569 tatatatttt aaggttgcac acagtacaca ccagaagatg tgaaattcat ttgtggcaat 2629 taagtggtcc caatgctcag cgcttaaaaa aacaaattgg acagctactt ctgggaaaaa 2689 caacatcatt ccaaaaagaa caataatgag agcaaatgca aaaataacca agtcctccga 2749 aggcatctca cggaaccgta gactaggaag tacgagcccc acagagcagg aagccgatgt 2809 gactgcatca tatatttaac aatgacaaga tgttccggcg tttatttctg cgttgggttt 2869 tcccttgcct tatgggctga agtgttctct aga 2902 5 23 DNA Artificial Sequence PCR Primer 5 cttggaggga agggagaaag tag 23 6 26 DNA Artificial Sequence PCR Primer 6 tgtgtatctt attgccagta ccacaa 26 7 27 DNA Artificial Sequence PCR Probe 7 ccgctgatga tatattcggg caggact 27 8 19 DNA Artificial Sequence PCR Primer 8 gaaggtgaag gtcggagtc 19 9 20 DNA Artificial Sequence PCR Primer 9 gaagatggtg atgggatttc 20 10 20 DNA Artificial Sequence PCR Probe 10 caagcttccc gttctcagcc 20 11 49001 DNA H. sapiens 11 caattcccgg ccccactgac tgaaactgag ccgtaggcga ttggtaccaa aatcaagtga 60 gaagccctcg ccgagaggcg tcgggagaag cgcgcagaag ccgggagctc taagggtgcg 120 gcggagtgc aagtcttgca cgcggggcag cgccgcatct gcggagaagg gacgccgagc 180 ggagcctgct gccttccgca ctgctcgagg gaaagccgcg ccctctgatt ccgccgaggg 240 ggaaggcgcc cccgggcgcg gagacaggtg gcccgctact cggccgtcgc tgtttccacg 300 tctgcaccgc gcccagaagg cctctcgccg ccccgggggc gccctgtgca cgcgctgagc 360 gcttctcctg cccggcttct gctccgctcc cgccgcccgc gggagccagg ggcggggctc 420 acctgtcagc ccgagccccg cctcgcggca cccaatgtcg ggaggggatg gcggacggac 480 ggaccggccg tccaaccagc gcgcggccgc ggagccgcgg gccaatgggc ttcgcgcggc 540 ggggcggggc cccgcgttta tagcgctctg acagcgcggc ggccgcgctg actctcctcg 600 gcggggaccg cggcgccggc cggagcgagg agctgcgcac gcagcgggtc ccggcgccct 660 ccccgcacac aaaggcccgc gcgcgtccgg agcccgcggc ggggaccgag ctccctcttt 720 cctgccctgg gggccgggag ccgcgcggac tgagaaggct cctgcgcgcc cggaggcgcc 780 ctactccgct ccgtgctccg ggacatggaa ccgcgccgac gcggcgcccg cgcgctcggg 840 ccgctgcccg ctgcactgga tctatagtca caggcggccc cgctcggggc gcagcgcccg 900 ccgcccgcgc cggtcgtctc cgctccgggt ctttgtgtcc cagcccgcac ccgccccgcg 960 cccacccgcc agccgcggcc ggcccggcac cctggagccg cacgggagtc ggccgtccga 1020 gctgcgtccg gcgcggcgcc ccggaacccc gagtccgccg cgccgccgcg ccccgcgcgt 1080 gcgctcccgc cccgcgccct gagggccccg gagagcgagc gcacctggcc cggcgaccgc 1140 tggagctgcg cagcgcgcct cggagctgcc tgcgggcgca cgccgtcttc cccgccagtc 1200 tgccccggag gattgggggt cccagcctgc gtcccgtcag tcccttcttg gcccggagtg 1260 cgcggagctg ggagtggctt cgccatggct gtgagaaggg actccgtgtg gaagtactgc 1320 tggggtgttt tgatggtttt atgcagaact gcgatttcca aatcgatagt tttagagcct 1380 atctattgga attcagtggc gtccgcgatc cccctatgtc cccgccccgg ggtccgccgc 1440 gccgtccggg cgggaggagg ggtcagtccg cggggcctcg gagcctgttt ctggaacctc 1500 ggttccccgt cccccacccc caacccccgc cccatttcac taggtggaga ctcctcgctc 1560 ggctttccaa cccgagcccc gctggaacgg acggtctctc cgcctttcct cccccgaacg 1620 ctcccaggcg ctaaaagcta ctatcggctc gggtgtcaag tccgggaagg tgtccgatgg 1680 cgatacctga ccctctcctg ttttcgagga cgaaggacat ggccacaatc taggctggcc 1740 ggcacgcggg gactggtggg ctctggagag aggcggagat gctgcattcg cggggagcgc 1800 gggcggcgtg gtccggggcc cgcgggcggg cgaccggggt ggcaggacgc tggcagcgaa 1860 gcgcgttctg gagaggggag cctggagtcg ctacgctgcc cgcagagccc tggagccggg 1920 gcgccttggc accgcgccgc cagcccgagg gtgcgcgggg agctcgcctg cttcgcagga 1980 gaactcgggc gtcgagccct ttcctccgcg ccggggagac gggccttagg cttctccctg 2040 agggcccgcc gcacctcggc ctcccgcttc gttcataagc cggtagcccc ggagtatgcg 2100 gtctcgatgg ccgacctgat tgtaatgcac ttcctataaa agcttagggc cctgcccagt 2160 cgacactgct cctgaagcct tctccctcgg gaccctggta ggaatgggat ccttaggatc 2220 agatttgctc ttaccggact ctacagccgg gagcgagcca ggccttgtgg agagtaactt 2280 tcagtttggg ccaccagagt gcattcagaa tttagaaaat cccatccatc cctaaatctg 2340 tgtggtcata actcgtagtc atctgggtat tcagtactgt gtatcccctt atttcgaatc 2400 acagccaaaa catattttac agaatcttgg aattgtagtc tcgggaaact tggagaagaa 2460 gtatgcagac attagctggt ttctggagaa aacgtttgag atcagaagca aaatcaatgg 2520 cctaattgaa gttgagcaag ttgggcctgg ttttaggaga aaagaaatgg gggattgatt 2580 tagaaatcac gtcttaaagg agtgtgtcca ttctcttaaa agtgtcaaat ttcaaattca 2640 ctaacatgtt aaccaagaat cccttcatga aaagggcgaa aacgtcggtt acaaatcggt 2700 ttaaacaaat gtttgtatga tgctagaagg cactttcaac accgctcata cggagaagtt 2760 acttagctct gcctccttcc atgtagtctg ctcttgcatg gattatattt ttaatgtaaa 2820 ttgttgtatt tgctgatgaa gtactggcgg cggcatcttt gcatcgatgc cggctcggga 2880 ggcgccaggt ggtgccggaa ggagccgggc taggacctcg cgcagcagcg ggtcccggag 2940 tccgggagag gcgggcgggc gggcgaggcg gtcgcgggga gcccgcggcg ccgctgcccg 3000 cccggtgcct ccagaggtca ctcttccatg cggaatcgcg cagcgccagg cctcgcccct 3060 cccccaggcc gcctgctcca gccactctgc actttcactg accggttctc tttgaggctg 3120 tttttttttt tcttatgagg atttaatatt tctgtttaaa tctagttgaa agcaattccg 3180 ttagcctctt cagcgtttag ttcggtgtgt gtatctttat ctttgcgcta tattaactat 3240 tagtttgtgt gtatccggta ggagaattag aaatacctag ttgggagaaa aagaaaagta 3300 gaacaatagt tatttcaacc taaggtttag acgttaataa cttctttttg taatgtgtcg 3360 agatgggggg tcctgggggg aggtgacagg tactcaccac tccccccccc cattctgatg 3420 atgaagatga gtctgtcttt ccagctatgt ccagacctgc gagggccctg cgtttctgga 3480 agcctgccgt ttgcgcggtt gaggttgctg ctgctgtctt gtcctccaca gcagcatttc 3540 ttttaaaatt ctcctgataa cggcctgcct ggatgactgg ataatgtgtg cctggaaaag 3600 gtctcccttg cagctgaatg ctagctccag agatcagaaa gatttcttcc tgtaggagcc 3660 ataggaaaga gtcctctcta agtttttgag aatgcataca accccctgat gacagggggt 3720 cgctttcctt ggggaagttt tatatttatt tccagaggaa agtttgaatc ggtaaatatg 3780 atgtggcagg aaggtaatca aatgcattga agtttcacat cagttcctat gaactgtgga 3840 acaattcatt tgtaatgaag ccgccatcag taattagatt tgtttcattc agaggtcagc 3900 ttttttagca ggtggtcgac acagggagca tgcagcagct gtttggatac agggtccaga 3960 aaaccctttg taaattcagc gtctccgtaa ctactttaat cacattgtcg gctctcccgt 4020 ccctgactgt atgtaataat ggaaagatgt cctgcgtgct gaaacagtag ctgccctgtt 4080 aggttattca cattgctttg atacgttctg gtagagttgg gtccgttgta gccattttgg 4140 ttgtttaaag ttttggtttt ttttttgttt tttttttaat tcagcagaga acagtaatgc 4200 ctagcttccg tttttaactt aacacttcag tagaacattt tcttccaaga gggagatttt 4260 ggcctaagta aagtagtggg ctctttttta aaaaaaaatt aattttactt taatgtgagc 4320 aaatctgtat tggtatggtg ttctgcaatg cattacactg actttgaaaa tttcgagtac 4380 taatgcctta tgtctggggt taccattccc tgtgcatcac atactagtta gttaacatag 4440 cattttgctt ttcccatgta attttttccc tatataatac tggattcctg atactaattg 4500 acttgataca aaagaatggc tggatgatat ccagataacg tataatacat gggcttcacc 4560 acaatcaggc tctgaataaa tacagacctg tcagagattg ataaaataaa ctacaatgga 4620 tagtgctgtt taaacagtcc attcaataac atatataagc cagcctgcct tccattgtgt 4680 ctgaaattct tatttttgta ggtaaacaaa tgcacattca gcactgattg aatagcccct 4740 tgaactatgc tccacagttt gcgtttgggt taatcttgtc ggttttaata tagagagaaa 4800 aaagctcaaa gcaccagggg tggaattgtt agtgctttca catccacatt cctcacattt 4860 tgtcaggatg ataaactgta ggtaatggac tgtcgttgtt ctgcaggaca actgagccag 4920 gcagagcaca aagactaagc taaagcgata cctcacaaca tgcttggtag ccttcttttc 4980 agatgagaat ttatttgaga atcatgtgtc tagggactgc acatcttaac ctcaacagtt 5040 acagcttcaa gccccagaaa caggagctgg aggttaagat gatttgctaa gcacctggtt 5100 ctaaatcttt tacaaagcat aagctgttga cgctggttct gccgacgcaa agacatgcag 5160 atgactccaa catttccaga ggcttctgac ttaagctaaa gtgtgtggac aggtgaattc 5220 gccatgggcc tggagaccag cttgctaaaa actatgtgtt tgaatggttc ctccagacag 5280 agtcagctga agaacaattg gtggatttat attaaaacct cttgtctgta aacttactga 5340 ggtgcatcct tcggttggtg gatcagtgag ataattgcct tcagatggac attgcaactg 5400 gagcaactaa atccttgctg tctttccttc ctctgaaatc ttccaggtag ctcccgagag 5460 cttcagtatg acaccaaact tcgggcgacg ttttagagtg cgttcaccta atgggaaact 5520 attcgagatc ccagcgtgac tgcagtaatg cgtcatagga atgggagtgg caggggaaaa 5580 ggaaatacag attgtagacc ctaataaaaa aatttttagg aaagatattt ctttaacgtt 5640 ttatgagaac ttcattctta aaatacttaa ttgcaaatta gacaaataga agtgctcttc 5700 taaggaaggt gattaaactg gtcctcctat cagcctaatc tctgcctgcc tttgctgctg 5760 acataaagaa cctgtttttc aggtcactta atatacatct acatagattt gcttatgagc 5820 tcaccctttg tgtagcggag tagagcctta aagaggagtg ctcaactgtt taaaatattt 5880 tgattaaaat atgcagaacc catagaacta taagcttcta gtcaggaatt agctctttca 5940 gggaacagct ccccccttct ttttaagggg ggaattagaa ggaggctggg ggaggaatat 6000 aagaacagca aagaaggaag gatagcaaat gggacatgtt ccgaacagct tggaaaaact 6060 cctgtggctt cattgtctct ataaagccaa agaatacaaa gacataagca attcagccct 6120 tctcccatga tggaagatgt aaaccgttga catgcctccc ctgtttaact tgtttaattc 6180 tcattttaaa ttcagcacga tactagccgt gtgaactctg aagatttctt tagtaatcca 6240 ttttgtagtt ccgaatcaaa aacaaagtga aagggtctga cacaatttgc ttttattttt 6300 aggcaaatca accctggtca tagttaataa ggggattaca actcagacta ggtctttaca 6360 gatgtgatgt aaatcaaggg cagagtataa agaaactgat cccttttgat tgaagtatag 6420 taaaaaggca tagagaaact agcagcagta atctgattgt atggcaataa aaccaccatt 6480 ttctgtcttt cagataaaaa taatgtggta aatccatgca gttcataaga tgtaaaggca 6540 gataaagggt gaagccatgg caacatatag attagcttga tgttagaaat gacacgtctc 6600 tgaaaagggc gcgggacgaa ggcccttgcc tccaggctgt tgggcattat gtgagaacca 6660 cacagacttg gaaactggga ttaggaagta tgaaagctct acttgtggtc tgggatggct 6720 gaggcagtaa agaaaagctg ctcagttctt gctcattggt ggtggataat atggcaaagg 6780 tagatttcat tgactgcctt ttttatagat tgagattggg gctgattaaa acttcagatc 6840 actgcagttg ttagggcctg ggagattttc ctttttaact cctggcctaa cagcagcagc 6900 cgttctgtag gattaactgc acttcgcggt cgttgcctta atctatttgg gcttcaggca 6960 gggacatgct gggaaggaac agagaccaga ggggataggt agggctgggg ttatctgaaa 7020 agaaaacaga gaccttttga tttcagccat cttttcagac ccagctccct ctcccgctgc 7080 atgggagaag caaaggtaaa caggacacat tgtccctctc cctcagccac agagctcttc 7140 tgtgagtttt gtctttccca ccctggaaaa aaagataaaa tacaattttt aaaaggggag 7200 ggaggaattt agttttaatt caaatgagta gtaatccaat atgccaaaag cagtgggctc 7260 tacctagatg taattttact cgtaaatgtg agtcttaaac tttgagttga atggggcagg 7320 ctgttagagg tggtgtaaat tacaggatta taaaaatgtt agtgctgccc agccttaaag 7380 tcaaaaacag aaaaatctct gtgctgttga gtcttcccgc cctctctcct gaacaacctt 7440 gtaagtaagc tagacttttg tttttgcctt ccatactttc catttcagcc attaaacaaa 7500 ataagccatt gaaaccacga ttgggttcca tgcagagtga catccgcaat cgggtcaagc 7560 cagaaggaaa tacttgctcg attgccccct atttggcatt acaggaaagt ctccacactt 7620 tggaagagtc tgaactctca agacattgaa aatgccaaag gctgcaaaca ccctgtgtct 7680 ttcttgatgg agtgcatctt ggtgtgtttt acaaagggga attcagtgct gtttttttgt 7740 tgttgttgtt gttttttttt tttaaagagc agcatagggc ccttctagac tcttggattc 7800 tgtgtctgac aaaaatggtc attaaatgag caatattata atttagaccc atttcactga 7860 ttttgttcca aattctcaac tgacttgagc atctgtttgg ggctgtagat acattgccct 7920 tgttgactgt ttttctcgtt tctatgggaa ttactgtagc cattactatg tagctttcat 7980 agactcaaaa catttttaaa gtattgcata taggctggcc atatccagtg cctgttactt 8040 taccttcttt ttctaactta atgcagcagt ctgtattaac agatccattt catttgtcta 8100 gcttcatcag agagaggcta ccccctgatt tacaggctgc tcacatccaa gcaccttgca 8160 ttctacactt gacagtgatt gctaatggcc cattcaacta aagtatttgc ttgttaacag 8220 ggaacagaac atgataaatg tccagcaagc ttgctgcctc cttcagcttt tcaaacgcag 8280 actggtgcat atttatggca ggcaaatgac aaaagaaaaa gctgaattgc cctggcctcc 8340 agctttctat cagaaacagg gttaaagtga ttaaagcaat cattcaagaa agccctgccg 8400 tttgtttact aaccttcatc caacatttag ctttgtagtc tacctgtgag aagatatttc 8460 agaagtatta gagataagga aggaggatct agcaaaccag tgaaaagagt aggtgaccag 8520 ttataaaatg ctttccatgc acattgaatg ccaggcgaac ctatttctgt tattccagca 8580 gacaatcagc agtggctcta gattattaac atattttcct ttcatgtata aattcaaata 8640 tgtaattcta gtccaaagca ttctgtggct ggtaagcaca tacttgctga tttcaaataa 8700 gaaaacatag caagggaaag ctccattaaa caagttgttt ctgcccttag taattctcta 8760 aacaagatag gaagaaaaag tggacagtag tggagtatta atagtgtgct cttttcattc 8820 tctaaagcac gagtaagtaa gcgttcaaac tactctgtgg tgggcataca tttagagcgc 8880 tgtgaatgaa ccactgctgt tctgccatac ttaatttatt tatattatta tttttatttt 8940 attgttgttt ttatgtatta ttataattat ttatttatat tactaattta ttttctcaat 9000 ttaaatcctg ttgcatccaa ttttaattac agtttttgta tctgccttcc catacttgct 9060 acccacgtcc ccattgccac tgcggcctta tccatgtttt ctgtgtacac cactctcgta 9120 tcaccccaga ataattatga gtgctaccca gacttttgaa accactagag tcaacatgtt 9180 tgtctttgag gaaagccaat gatgctttag catttttggc aggggtggat gtgtgtttaa 9240 gtggggtggg tgcagctcct tattgtctgc ctattctact gttgttccca atccacattc 9300 cctgcggggc acctaacctg tgtgcatagc aaagaatttc cgaccttcag agccagaagt 9360 gtttctcaat tgatctcttc cagcctaggg ttatagctga tgaattataa tccttgctct 9420 ttccacacct ttacctgggc ttaccatggc cctaaaacat ttgcccagaa tcagaattgt 9480 ctcatgagtg agtggggcaa ggcaaatcct gttccagacc agctgagaat gtacctagct 9540 gcagaagaag ttagaaagtg tcatctttta cttatctacc agaactatat tcgaggtaca 9600 ttttagattt aaaaaaaaag caagttctcg taggccttga atccccccct tgctatggga 9660 aaatggatca ttattataat ggactgtcca gtaaagttca tgatttctcc tagacatgtt 9720 ctctctcttt atgacctaga tcaagagtga tctctttaag tcttttcttc ataatcccac 9780 agcactttgt acttagatgt acttagaaag aaccatatac acggtacgtc atgattgata 9840 tgcaagcctt caccactcta cctgtcctaa aagtcaggga cacaccttct tcatttcatc 9900 agtccctact tctatccagc attggcatcc agtaagtatt agtggaatgg acagacaacc 9960 cgaatttgtg ctgatggcag tttaccctgt tttaactgtc atccttctgc tactagacat 10020 ggatgagacc tgagacgatg ggactgctca gaggtccctg gctcttgaac tttagggcac 10080 cagaatcccc tgcagggctt gagaaaacag gggtttctgg gccccacccc cagagttcct 10140 gattcctgag gtctggggtg gggcttgaag atggacatgt ttaacaagct cccaggtgac 10200 gctggcaact gctgcctcag ggccatgctg agaaccctcg ccctacacaa acctttctgg 10260 gaaaacaact caacattaaa gctgtttggg gatctctgaa gaaatctgta gtccttgcct 10320 tgttggggga gcatcaggga tctaaccatt gatggtggag tatttgttgt taattcagca 10380 agcaactatt aagtgttagg cctgttactc ggctctaaca atacaaggca gagtgacctg 10440 taccctcgag atttaaagtc taagtcctgt agagagaagc ccaggtggga gcaagcacat 10500 ttagagttag gtgcttggtg caaggtgggg acacagaaga agggaatggc atttgcctct 10560 ggaggggtcc ggaaacagcc tagggaggag gagcttgagt cttgaaatac tgtgggcatc 10620 tctaagcaaa gtcacagtag acagctgaaa taaagaaaat agtaagcaag ccaaagaaac 10680 agtatttcag ccaagggcag cgtgtgtcta tcacgtccac ctgtgaacac gtcccaggat 10740 tctctgcatc cggccattgc tcaagacaga tccctcacag gaacagctaa gccactgatt 10800 tcagctacct gttcacgtga gaattatcag tacctactgc ttttcaaaat gagtatgatc 10860 atggataggt gaggcaattc agtttcgcag agacagtagg gcaagtgcca ctgtagttta 10920 gttaagggca catgctttag agtttggcta tgtgagtcca atcccagttt agccatttat 10980 tagctgggta gctttaggag cagtagcctt agtgtctctc agttgtccca tctctataat 11040 agggacaata acataatagt gctgaataaa agagtaacaa aattttggtc aacatttaat 11100 gtatttaaag agctaagctc cgtgattggc acaatgaacc aatcaatcaa acaccagttg 11160 ttattaataa aagtcagttg aatatgtact gtgtgcctgg ccgtggttca atttgccttt 11220 gcatacaagg aaaaaattaa aatactctgt taataaagac tatagcataa tactttcacc 11280 ttaaacttct tgatgttaat ttattttgtt tacctgccaa acttctactc attccttatg 11340 actttctgct acatgaaaca ccctttgtaa ttcttttgtc ctattaaatt aagttctctc 11400 tcctctgctt tcctgctttt ggtgctttct aataacactt ttaaccctgg actttctcat 11460 tcagctgtgc aactgtggac tgagaggagg ctctttgaat tcattttgta tattctagta 11520 gagagtactg tgagcagttg ggttgttgaa tgaatacatt aattcaacct ggagggatgg 11580 gcagtattgc attttttaca ttgatattac atgatattta gaaaactgct taactggtgg 11640 acgttgtttt attaacagca ttttgtgtat agcactcact atgtgccagc tgctattcta 11700 actgcctgac aaatactcct gaaaccttca tggtaaccat atgagggaag cacttttaat 11760 atatccataa taccaacggg gagactgtgg ccaaattggt taattaactt agccaaagtc 11820 atattgaact aataagtgga tttaaaccca gctagtctgg ggccagggtc cctcttttaa 11880 tcttctgcct cctgcttatg ctgttgcatg gagtagtctt tatcatataa ctaaattaag 11940 catgcatttg cttaaagcag tgcatacatg atggatcaaa aagtttgtgg tataattggt 12000 ttaattctgt cattatccat tttgatttat agtcactttc ttatgatggt cgtgtagttt 12060 taaatggaac ctttgaatct ttgatataat aaggttatgt caaatcttgg gtataataag 12120 gttataccca atggaaacag aataatgatc agcccattta aaggatgact ggagagttat 12180 tacaatacat aatagtcatg catatattga gtagtattcc tttggtaaca ttttcctttt 12240 aaaaattgta acatttgatt gttccttgtt gggagaaaag gaggtcagat ttttgagggg 12300 agatccattt ggtgagatgc tgagtgtgtg tcaagctaag gagatagtat gacatctttt 12360 ttagagtcta gtcacaatta aatgccattt tattttggat tttgggatcc gtgccagctt 12420 ccagcttgtc agagctgaga agactcaaat caagtccagg cttatttcta cagcaaactg 12480 ggattctggc ttcttgccgg tggattcatt cagtacagcc catctggctt ttgatgttct 12540 gcaagtttgg agccatttgt tgaaggaagc caggcggtga atattggtgg tcctggggtt 12600 ctcttgactc caagtggtgc cccttggttt gcattttcac catgcttagc atctgcttac 12660 ctggagacca tgcagccgcc ggccagaggt ctccaacaac caaatcttca tgccttttag 12720 aactcagagt ccccagcaca tcctccttcc tcctccttgt ccaattactt tcatgcagtt 12780 ctcagtagct gcttgtttga atcacttata gtatttaact tctagggtgt ttttgggttt 12840 tggtcaaggt aattccaggc tgaatgtggt gactaagcag gaaataaatg ggtcgtcctc 12900 aaagttacag tggagcgctg tttctatttt cctaaggtac acagttgtgg gggcgatccg 12960 tatggaagtc aggaacccag tctgattttg cttccttttg atggtagcag tacagacctg 13020 gctgttttgt agcctgcttt gtttttcttc cttttcttcc ctaacttcac gggctgtggc 13080 aaagccctga gacgtgcagg aaaatgtctc ctgtcatacg cccacagcag acctagccct 13140 gaccctcctc tgaagcccag gaaggaggta tctgtgaagc agcctgcttg taaagcaatt 13200 gcacacagcc ttgtaaactg tgttactggg ctgattatac ttgattggca aggtgaatct 13260 cttatagcaa aagagaactt ggagagtttt atctcatctt atgccttatt aatttgttca 13320 ttctttaatt acacagccac ctattgagca ccctatttat gcaaggtacc tggtcggggg 13380 tcagagggag ggtcccatgg taaacgagac agactcaatc ctggaggagc aggaatggca 13440 gcccctcgct gggctgttgg ccccaccaaa agggaaaggt ttcattttaa taatacatgg 13500 gtgaatcatt tttgtcaata ggcaaaattc tttgtagtta aaaaaaaata tgatggtagg 13560 aaggaaaggg atgggcagag ggttaaaaca aaagatatgc tctccctaac tctagattgt 13620 agtattgtta tgcttgtcac tgtagctgaa ttccatttct ttgagttttt tcaatgccaa 13680 ggcattccct gtatgactta cgtgagcctt tcatctccgc gatttttccc attcaggtaa 13740 atgagcaaat ggatttgaac actcatatct aaaacaagag agaaccagct ggaaatgccc 13800 tttgaatttc tttctctatg taaaccattt ttctttctgg tgcctcacct ataaataaca 13860 ggagttccac cttcctttat agactcttgc tgaaagcatg gtttggaaca agaccgtaca 13920 ggtgcacaca aattacagtt gggaaagaag cctgcagtgc atcttgtctc tgaaggttat 13980 gaaatcctcc ttttagtaat ggagctggcg tgatcaagcc agcaggatga aatttggcat 14040 ttgtgagatc accccccttc tcacttgccc actgtacata gcatcccagc cttactcttc 14100 aaatctccac attttttctt atctagctac aaaattcata ggctgatttt tttggggtgc 14160 gtgtgtggtt ttttttttgt ttttttggta aataaagacc tgcattttta ttttgatata 14220 ggtggttgag ttttgtcttt aatttcatga cagagattta actagtctca acttttgaaa 14280 agacaacaat gatatttggg gatcacacac ttaaagttag atttctagat gattaatacc 14340 aaagtagatg attttttagc ctcagccatt tataggtatg cccttctgtg aattttttat 14400 gacagtgaaa atcatggcac agataaaaat taaataaata cttctgttat tttcctgaag 14460 aaaaaaaaaa aaagcttaaa ctatgagaat actgtctttg agcactttaa aataaaattg 14520 acttcagcca gcaggatttt gagcattaca tcacaaataa aaaacaagat taacatcaaa 14580 aggagtcagt tttcattcaa ttgtgcagca ctgtgggctg tgaaatttaa tattattttg 14640 actcatatgc taattgtaga ctgacagagg aaaatggatt gtgtttaaat aaaaggatac 14700 acagcatcac acgcagctgt atcaaataca agttgaggtc tttgggccag gaactggggg 14760 ccctctagct ctgttattgc agattcaagt ttgacaaata aaactttcct ttagactgta 14820 gtttaattac tttttttcaa aggtatgcgt gatgaagagg cacaaataca cctcaccttg 14880 aagagttgct aaactggttt gtgtgccgat cagttcaccg tgtgtttgaa tttctgtgct 14940 tctcatcttt ccttttcttg aaaagatttt gcttgtcatt ggtgtgaatt gtacccccca 15000 cccccaccca tctagtcttt gctctcagat ttataacact ttaatggttc caaattgtat 15060 agcctgctct tagacccctt ttcttttcct tgaataaatc aggttcatgt tgcagacgat 15120 atttgtttta ggaaagtgtg aaagaagggg cacctgtgaa aacacgcaat tgttccaaca 15180 cacatataca tccaaattaa agcagaaaat gtcaaagcct ccaatcacta ccttatttct 15240 tggaggttta aagccgctga gaagatagtg gtgccctcgc tggaagtttt aaggtaatta 15300 ctttttactc taagcagtag tatctggtaa cctaattccg tataaacctg acaccctatc 15360 gctacacccc agtatttctc tgatttcaga ataagtctgc gtagaaactt gttctgatgt 15420 taaagtgcaa aagggggcag taaagtgcta tccacaaaaa aggaaaaaca ttttccaagt 15480 atttcttatt actgcctgtg tctttcgtag gccctgcctt tatttattca ttttataaca 15540 aaactcttat gtttggggca ttcagagaat accttattaa gctgttgcag caatctagca 15600 ttaaatggaa gacatgcaag actgaagatc ctgcctgttt atgaagtgtg ccatcaaatt 15660 cacatgctca tgatgcagag tccttctttg ggagtattcg tattcccaag tgcacagagc 15720 acttcggaaa ggagccttgg tctttggtgt taatgctctc ctagctccgt atagatgtgg 15780 caggcccaaa gtacatggtg gggtgaaggg tcaagggttt gggcttatcc agagcagcgt 15840 gcatcctttg tcaggaggtg actggaaaca ccagccaatt acagcagaac tgcagactgc 15900 tcatctgcat tcggaattgc agatgaacca gtttgtactc gacttctctt cttcactgta 15960 ggctttgaca tttaattaaa aattaaagcc ttttatggaa aaagtacatg ttttccaaaa 16020 tggggtaaat tcgaagtata cttgatacag aacactggct tgggaataaa cctgtgatat 16080 tacatgactt ttggtttgca actgctaggc tgagcctctt tgtaaagctg ggatttagaa 16140 tctttgaaat gtttgtacag ttcaatgatt aagcataaat tgtatatatt cccttttttt 16200 cacttatttg agtaaacaag tttgttacta cagcttctgt ggactcagag atttatgtat 16260 taaataggcc acaacttcaa ctaggataat tttatttatc tgcttgttag ggaattgcat 16320 caaaagttta agtctgtagg cattaaatat tttaaatgct tatttttaaa gtcaattatg 16380 aaagatagca caaagttttt ctgaaactac attaaaaaaa taatgtttta atcttatcac 16440 aaaagcattg actatttatt gcaaagaaaa cacagaaagc taaaaatcat tctaagtcca 16500 ccattcagta gcccaaagtg gtctcaggta aaggcggtgt gtgtgaccat ttgtttatgg 16560 ttgtctccgt gcagtcagca aaataaacag aacaacatgc catatattat tgatgtgtat 16620 attttcaact gaaattagcc atctgcttac aatgatcata tacactaatg gtataatttt 16680 gaaatgaaaa gaaaaataaa ataattcttt gtggagagta atgcgaattg acttatgaat 16740 ctcgccctgc ttggcagttt gctctagagg tagaagagct ttatgtgtgg gcctcctccc 16800 cccccacaca tttattctgc tcacacttgc accagcatcc atgtcaggac tcaccttgtc 16860 ctgttacatg agtaacatgg ccctgattct caagtgcatg ataactgcca taattacaca 16920 taaatattaa atatttaaat agatctttac gtgtgtaata ttaggtagaa gtggctctgg 16980 atcgaatctg atgcttttta aatagaagct ttcccacaac atttccaagc actgtcatcg 17040 tgtctgtctc gatttggggt ttacctggcc tagttatctg tctgggtgta gaaactggta 17100 gttcctgttt gtatcttttt tgttctgatc tctttattct gtgtcagcta aatattcttg 17160 cagtcagtta ctaacatatt aactcatcct tgtttggaaa ctttggcata tccttccatg 17220 gtttccttcc gtggacctgt cgcgtctctc aggagagcca ccaggtatat tgtcacacat 17280 ttcgcatgta ttttcagaga ctacagcagc atcaagtggc cccccagcga tttgggtttt 17340 cttctcggtt aatctacact ctttggccaa ccgtgagaaa acttgtaaga aggcatcaga 17400 tgtttgtgct aaggtgcgtg tagtatggtc agaggaagaa agaagcaggg aaaatggagt 17460 ggccgtgggt gggaggggaa gcagggagtg caatttcggg ttcactacac agctctccat 17520 aaacttctcc actgctggct tcccacggat cctcctatta cactgggcaa agtgcagaaa 17580 tagatcaggc gaccactgcc tccgtccatt tcccaggcac cctgtgagac ccgataatgc 17640 aatacaggtc agcagaaaag tccagacttg acatcccaac gtgccatggt ctggtctgtg 17700 aatgaaaatc acatgaggtg acctctgaac tctaagtggc tggtttatgt tttcagtgta 17760 ttaggcccgt gttttaaaca agcatgtgct cgtagtgtag gttaaaactt tctgttgtct 17820 tcattaatta tgctgtgttc tagtctatta atattaaaga atattgtgtt gcataatgac 17880 taattttttt attttttgga gacggagtct tgctctgtca cccaggctgg agtgcagtag 17940 tgcgatctcg gctcactgca acctccgcct ctcggattca agcaattctc tgtctcagcc 18000 tccgagtaac taggactaca ggcgcccgcc accatgccca gctaagtgtt gtatttttaa 18060 tagagacggg gttttaccat cttggccagg ctggtcttga actcctgacc tcgtgatcca 18120 cccgcctcag cctcccaaag tgctgggatt ataggcgtga gccaccacgc ctggcaacat 18180 aaggactatt ttttaaagtt tttacaatta tgactgtgaa gttgaaatgt ctaaattatt 18240 agagatccag tttagattac taaatattta tgtctaattg agatgattag acttagccaa 18300 agtatccatg tagaagtatt agagtctaga ttggtgaaaa acttgaaaaa gcttggctta 18360 agttcaatag gtaatccaag agtaaaaaca gattccaata tcagatcttt tcaccatagt 18420 catgttaagt ttggaagccc tacttgagtg tttccagttt tttccacatt atattgtgtc 18480 tatatttgat tcaaaggcag ggcatctatt gtcttgctta ggactgattc actgggaaaa 18540 gccactggag ttgcctattt ccactcagta tgcctcactc ttagagtagc ttcccatggt 18600 tcccaggcag gccctccagt gagaatgcac caagccacac gccatggcct gggaagcagt 18660 cctgaacctg gagattgtct tgatggaaag gaagaggcag ccttcccctc ccaggaagat 18720 agtagagagc ctgctctgac ttcgctcagg gatggaactg gtctggctca gttctctctc 18780 ctgtgtggga catgaatcac tcttggtggt ctttgctttt tatttgggct taaaatcagc 18840 agactttatt aaatgacacc tctctctaac cactctctgt ctgggcgaag tttaacaaga 18900 acagcctccc cccatgtggt atgggttgta actgtggcgg tttccctctg ctgtttttgg 18960 ttacaagatg aacattatct gaacacacag aaagaaatct gtatttggca tccataatgg 19020 aaagtcagtt tagtaattta aacttagcca gttatcatca tcataattct ttttaacact 19080 ttcaaagtca gcataggaga agtgtattgt tgaatattac aaaatattta gggcatagat 19140 agatgtgctg tgtagtttga tttgttaatg tgtctaagca atcaaagcaa cagaattcaa 19200 atataaaccc catcacttcc aaaataggaa ctctgtttac tgacttgatt ataacatatg 19260 gaactcaatt gttttccatt aaaaaatgat actattagga aactcacccc attttctttt 19320 catatatatt ctgctatttg cataattgtc tggagtccat atgtaatatt aaatgtaaaa 19380 cacaaatgcc atgtagctgg tctgtttctt cctcaccttt tggttcctgg cctcctgggg 19440 aagggttgca catctgagcc gtggtctcag atgactgcct cggaagaagc ctcttccctt 19500 caggcaccac tgatgtgtgc ttggtgtgga gctagacttt ccctggctct ccatgtgacg 19560 ctcacatgtg cgtgtcttga tttcccttaa cttcatggct tatctatgaa cagcttgatt 19620 tgggggaaaa aaatgtgttt cccaatgctg gagttataat tgaatgtgct gcagtcaaaa 19680 ctgaaatgtg tgcagagaaa gggggctttt cctgtcatgc tcattgggca ccagtgtgtc 19740 ttcacctgtt ttgtgtgtta ggtccatgcg tcatgctgaa atgaagaaca tgggatgtat 19800 ggggctttgg acagtgctga gccaaaagca agtgctcaaa agcagctgtg tttgtattat 19860 tagtggttct ggaggtggct gattgccttg cattttaagt agagagggat tgtagaagac 19920 tgccaatact tagaactttt tccagagagg aagggtcaga aactgcatct gcagggctcc 19980 ttgctctcca gaaatgccag tgtgcctggg agggcatctt cagaaatcca gtctctcctc 20040 ctcagtgtgt cctgtaccga ctcagtggtt ctgtcttcag aattcctatc atgtctgtga 20100 tctgcaaata gtggtattta atttgacttc aatttgtata aatgttagct tctatttgtt 20160 cattcctatt ttttgttcaa ttaatacatt atttattgag catctactct gtgtcagccc 20220 cttgggtgtt taatactgaa ttagtcacat gtgggacttg cctgccctca gggagctaga 20280 ctataaattc ctaatgatca gtggtctcca cttttctgtc actcataatg tctggcacaa 20340 cataggttac ttgagttgtt acactcacag tactgttgtt tgctgccatg gtgctttagg 20400 aagtgtgaga gttcccggga ggcagagtca ataatgcaga ctacacgtag tgaaaacatg 20460 gccaggagag ctgtagttca ggctctcagc tcaactgcac tctgtccact gagaagccat 20520 aatttcttca cttaaagtga ctgtgcgcta tggctgttta tatatacgct taaaaagtaa 20580 aagctgctaa accactcaag gattggggcc ttttgtattg atttaattaa aggaacaatc 20640 attgttttaa tgagctctag aaacaattac ttttgaagag ccgaggatca aattcttgcc 20700 tcacgttttg ccacagtgtg ttctgaaagg tgaattaatg cttttggaat catcaggaat 20760 agtgagcttt gtcacgattt actttttaca agcgtatcta atatgcatat tgaaatgtga 20820 gcctccccac cacacttccg ctttgataag catcccccgg attgccgtca ctgaccatta 20880 tagattttta acaaagttgg acagtacaca ctgaatgaaa actttacatc aaggaaggcc 20940 tggcgtgttt gtaaaatgaa ttaaaaggct cattaaatga tttatatgac ttacgccttc 21000 tgaaaatatg gcctcaaaca cagagatccc caaagccaca ccgacccctg cgtcccatgt 21060 tctcgacctc accgcatcag caccagcaag acctgtcgct gagacggtga gtgatgagag 21120 tcaagaggag tgacttgcat ggcctgggag gaaacctcct gtgaatcttt agttaagcag 21180 gaaaaaaaaa atcctcatga aggaaacagg atcttgggag cattttgaat gaagaaggag 21240 cttagtgagc caaacttgag acatagggtg taatgtggga gagttttaag atttgcagag 21300 atgtacagct tgggaggggg tgtaatgcat tttcttaaaa gagctgaatg aatggttgag 21360 gaaatgggta catctggttt ggttaaggat cctaatctct gaagcctggg atgcccccag 21420 ggcttgtaat ttaggaatac ttcccctaat agtagctaac ccttatatag tgctgtctgt 21480 gcaggctaca aaaggagcag attaaggata gaaaaggttt ggagtgtatg agaaacccta 21540 ggcaggaatt gactcctggt gtttgtaaac cttaaagatg tcctaaaaag gtcaaggaat 21600 aagacaggag aaaaaggaaa tgtcaggaag atgatcaatt taatgtttat ggaatttagt 21660 ttgtacttac tgcccggcat cttgcctgag gtttttaacc tcagcagcac atcagaatta 21720 ctgtgtgtgt gttggagggg ctgggggaga taaagaaatt agcctcatcc caaacattct 21780 gattcagtct gttacttgag aaactgaatt gtgttttgtc cataaagaag atgaaattgt 21840 ctacagagaa cacattgcca ttcacaaggt tgaggggata ccacagagag gctcccactg 21900 tgatttgcat ttgtcaaaag ttctagagaa ttcttcaaca gtacacacat ggttgtttta 21960 aatatatcat tgttataaaa attcgttttg agttctgttt cacagaaagt ttttttgaat 22020 gaatgaatgt catatatcct tgctaaagga gctcagttaa aaaaaaaggg accatccttc 22080 tcttttgggg gttgtacagt aacacattcc caagaaagag gtaacagcca catacatttt 22140 tcttcccaat aaagagtgtg ggtttttaat atgaatccat agtatgattt ctgttatgtt 22200 ttgtgctgct tcataaccac actcatgcac ttttcagaaa attaatacca ttcattagca 22260 taaatcataa actattccct tggtatgggt ttgaaattgg gggtgcccta tcatccttgc 22320 tttatctctt agtgaattat gaccctgtag tcatcatggc tggtgggcgt ctctggttaa 22380 agaaagggtt ggattggaag gattcagagg cgattctttg ttcttaggct ttaatatttt 22440 aatgagcctg caggcttggc tgcttacgaa cgagctgaga tttctaagtg tgttgttagt 22500 gttagcactt gtagaaggat gttcattagg aagttcttgt ttcagttttt cagagaaact 22560 ccccattaag aaagatcatt caggaacatg gctaccaaga aagaggaaag ggaggaggga 22620 ggctttcagc tataagcatt aaggggatat tgtatcagta gtcttagttc taaagatttg 22680 cttctgagaa ttaattggag caaatacatc tcaagggaag aaaaaaaaag atttataggg 22740 cagggacagt agttgtcctt gcaagtagag gacacttcat tttgcagctg aatcaatacc 22800 acaactaatt atttctggtt atcttttacg catttgtaag acattgcttt tgttcagtgt 22860 aataaaaaac ccattgtttg atcagtgact gactaattat gataagtaat ttgaaacatt 22920 cttgatgaaa cttgtctgtt aattaacatc aacagcacag ggaaactaac aggacaacaa 22980 agtattagtg gatccactgt tccctccaat tgacgagctt tctctgtggc atgcccaata 23040 aactaaagct gccaatggtt aaaaaataac aaacatgtgg gagatctgac tcaccacgga 23100 ggaagagtta tggtaaagtt acacaaagga gtactgaaat attacaagcg agggggtggt 23160 aaagaaatgt cagcaggtag cctgatccta cagcttagag taaggaaagt ggtttctttc 23220 tgtctttcct ttttctttta aagcttaatt ccaaaataca ttcatcccat attgatctga 23280 agtaagagac ttttgataaa ttaaagtgtg aatctgaaaa tgtgtagttt gggattatgg 23340 gcattgcctg gctatcttgt aactgtcatt aatactgtta atttttatca actcaatggc 23400 ttttttttct tatgctttta gatttctacc tggacaagga ctggtactat acccacagat 23460 aggagacaaa ttggatatta tttgccccaa agtggactct aaaactgttg gccagtatga 23520 atattataaa gtttatatgg ttgataaaga ccaagcagac agatgcacta ttaagaagga 23580 aaatacccct ctcctcaact gtgccaaacc agaccaagat atcaaattca ccatcaagtt 23640 tcaagaattc agccctaacc tctggggtct agaatttcag aagaacaaag attattacat 23700 tatatgtaag tataatttta ttcatttatt ttatagaaat taagataagc tatataggtt 23760 tgtatcaatt ttttgtttcc ttaaaattat tgtgacaaat aatttgatga aaatctatgt 23820 ggaaaaattg tccccccccc cttttttttt ttcaaagaaa acttcattga atttgggacc 23880 ctgtgctacc agtattcatt aagtatacat acccaaagag aaaaaaaaac actagaattc 23940 ttaatagtat tgaaataaat gtattatatg aatatattca gcatctctac tgacaaaacc 24000 atttttaagg accattggtg gattttgata ggtaaatctt gtgcattgcc ttttctcttc 24060 acccatccat ccattcattc actcattcat ttcgtattta ttctgtgcca gagactgtgc 24120 ttaagggcta gggattcagc agtgaaaggt ggtaaaatag catgttttcc tcaagaagtt 24180 aacagtctag agaagatgga gctcataaat tcgaaagatg gggatgacag gtcacattaa 24240 aaccagattc agaagaaaaa gacgaaactt ggtttgctta gtacattact cttttttgca 24300 tacatatata taatttgaca cgctgtttca agaagagatg gtacgtatcc cttgggtcat 24360 atctgaggct gacttgtgag gatgtgaagt cagctgatga gcacatttgg agcccacgcc 24420 tactatgtgc agatctctcg tcagcgtcat tcccagggcc ccaggtggtg ttaaagtcta 24480 ggtgactcag acagctgttc gcgtcattca agcaatgaag tcttttttct taatttcttt 24540 ggtttaaaat tatactcata attaattggg ttgaattttc cagtggcttg gttaccatag 24600 acttcagttt attagggaac tgctatctgc cactggttta ttatttgccc caaggtggac 24660 tctaaaactt taggtaggag actcttggtg atcaaactga aactcttgca tctcaaccta 24720 tgagccgcac tttattgtta ttttattttt ttagagacag ggtctagctt tgttgccgag 24780 gctggcgtgc agtggcatga tcacagctca ctgtagcctt gaactccagg gctcaagtga 24840 tcctcccacc tcagcctcca agtagctcgg actacaggca tgtgccactg cacccagctc 24900 aagagctaca cttcaaagca cagaatgaaa acctattttt aaagccaact tgatacatag 24960 agtagcttac caagaattag taacaacaac aacaagaaaa aaaagagaga atgtggtaga 25020 gtatatactt agtaaggagt aattattata aaataaaagc attctgaaat gaaacaggta 25080 gatggggtgg ccaagtatgc agcatagtag ggaaatcttt gaaaatgtaa aatagttacc 25140 aggtaaaata aatggaaact ttaagctttt ggaagcctaa caatgtattt atattagtaa 25200 agactttatt tttttatttt attttatttt atttttgaga cggagtctct ctctttcgtc 25260 aggctggagt gcagtggcgt gatctcggct cactgcaacc tccacctcct gggttcaagt 25320 gattctcctg cctcagcctc ccaagtagct gggactacag gtgtgcgcta atttttgtat 25380 ttttagtcaa gacggggttt caccatgttg gccaggatca tctggatctc ttgaccttgt 25440 gatccttccg ccttggcctc ccaaagtact gggattccag gcgtgagcca ccgcgcctgg 25500 ccttagtaaa gacttttaaa gtaagacttt ttcagtgaaa gctactgtta ggcatgacat 25560 ttacaggcaa ctgaaactga tcagatgcat ttattaagaa ggttaatgcc cctaggtggg 25620 gtgggagaaa gaaggtcgtg gtacgggaag aggggacaca ctagagatga gatgccctag 25680 ggcagtgaac gcatgtccct aatgcgtgga tgcagcccac gtccaccgat aatgccgaca 25740 cacccagagt ctctcttctt actttagctt atgacttcac gaagaatgct ttgcaaattc 25800 taagttcgca ctgggcgcaa gtggaatttt agtaaacatt aagagtttaa cctttagtgt 25860 gaaataatat gcaagatatg caaataattg tttaccaaca tctctttgct taatgtggtg 25920 agcatttaat aattgctttt tattaataca tgagagattt gtatttagaa gcagtttaat 25980 ttataattat aatattaatc tacacaataa cgacatctat tattttcttt ttttggaaac 26040 tcttcatacc acactaacag gttcattgca gttactgaac tactctggcc atcagagctc 26100 tccttagagt tacgatttac catgcaaaag catatggtag cctgggataa atgaatcttt 26160 cttaatacag aattgagggt ctcaagtttg aaactacgag aggctatttg aatgttgctt 26220 tgggggactg tcataagggc tgggtggagg actcagggct aagaagtttg ccaggaagtc 26280 cagttgagac tttcagcaga gttgaaagac ttccacgatg gcgtaggcag aggaaggcgt 26340 ttcagatact tgggaaaata tagaagccaa tttctcaccc accctacagc aaagctcatt 26400 gatctacaag tttccctaga aaggaaatgg gaaatgcaga gaacaaatgt taaaatagtt 26460 ttagaaatta atattgactt tgtattgctt ctgcataagt tccaagacac caaaacaatg 26520 aatggatttt aaaaagtcac tactttgcat atcagacaaa tgcacacaca cacacacaca 26580 cacacacaca cacacacaca cacacagtca agctctgtac tggctttttt gagaaggaaa 26640 gtgtttgaag ttagtaattt ttatatcagt acatttataa atagtgctag gtagcatgac 26700 ggaaagtatt aaaatttaca tgtatatttt taacacttca aatcgttggt tcactttgag 26760 acagtaaata atattagcat ttgagttcag ctttaataaa ttctacatgg gtttaacccc 26820 aaatctgagt gtctagttgg taagcgcctt cagaacgagc agtgttataa taaatatgtt 26880 attgtgtgct ggtttctttc catggagagg aaaaagagac ctgatgcttt ggaggagtgc 26940 ttgacttttc cccagtgagg agtagtccag agggactgac ttgcattggg gagtacccta 27000 catgaacagc atttcagaag aattaaacca ggaacctaga gtcctacttg ctagtcctgc 27060 ttcctaagct taatgagaaa gtcaatttta tttctttgaa ctttaattta tttccctaaa 27120 aaacgctttt agtattgtca ttgttctggc taatgatggc ggtctcctcc agtttcaagc 27180 caccttaggg ctgggcatac aaatgcaata taggatcact tgttagtgtg gtttcaaatg 27240 gacatgatcc tctgtaaatt ctttaaaaac atttaatttg atttgtggtg ttacctgctt 27300 taaaatatag tcatcacact tgtgagtttc agacgtgaat atgaattttt aatttgaact 27360 gtatttttaa acacactaag tattaactaa gtccccttag gagatatgtg gcaaactgat 27420 atgcatcctc attcattctt ctcatagatg gttatttgtt ttttaacttg tggcaaaatt 27480 atatatgaat ggtcaccgac ttaaaatagt tccacttaaa tttttcaact ttctgatggg 27540 tttattggag tattaaatgt attttcaatt taatgatatt ttcagcttac cttgtgctta 27600 tcaagtatca agacatagcc ccacctaagt catggagcat ctgtatatgg gtttttattc 27660 ttgtttagaa ttgacttttt caagtgacct atttcagtaa ttagccctgg gcctgatttg 27720 cataatgaga tctcctaatc ttcaagtaat gcaaagatgg agatattatg gccatgtggt 27780 ctgaagagac cttttcttta ttatgttcag atctttaatt gccttaaaaa tagagtagct 27840 aatttaccta acctctagtt attttattat tgtctttaaa gtttttttta atgttcatga 27900 aataactgtt ctgaaattgc ctattttcaa gggaagctgt gtcttagact tactaaatgc 27960 tccagttgat actgggaaag ccttcttgtg ttcgtagcct ttatccgtag agttttcttt 28020 gcagcatttt ctgtgcctgg tttagtttct tttcagaggc gacacccaga gctgaatgag 28080 tcagcaggtt tggtgtgtcg accctttgca acagctgtcc ttacgaaggt tctgtgggct 28140 ggttattcta ccttcgcata aaaccttgca aaataaccca caaagaggtt ttcgtcacac 28200 taccaaaatc atgtgagtca gagatggatg aaaaatgaat gccattgtgt tcatactttt 28260 ccagtgaaca gtagctacag cagagctgtt agacaaagaa aaccgtatta atgaagcgcc 28320 tcccaattta gcttcatatg gcttttgcat tattttgctg caaatccata gctaagacac 28380 atcttgtggc atagtccgta agtcatcttt ccgaaggact gtttgattaa aggttgttct 28440 gtgagatcca ccctgtgttg ttcatggcat cctcttggag gcctccctca ctctccatgc 28500 cttggcaaag tcttccttaa ggaacactga acaagtctgg agaagctgcc atttcttagg 28560 gccctcattg gttcagttgt ctatagcttt ttatttttta tttttttttt aataaagagt 28620 atgtaaaatt ggaaagcttc acaaacagct ttgctatttt ttagacatgt actccacttc 28680 taagcaaaat cacaaaataa agtaaaatgc ttccacaaat ataatgaaac aatattctta 28740 aagaatcaaa gcagaagaac ttcagagtct gttgcttatg ttaagcatat atttgttttc 28800 ttctctgctt ttgatttact tatttctggg gtgtaggttt ggcaagtagt actgaaacgt 28860 actgaatgca ctgttcttta gcaagatagt tacaggagct ttcaaatgtc ctcttaacat 28920 atagatttct tttagaatat agaataatgt gtgggctgta taaagcgatt atgtgcttta 28980 tttgatgaat tatttatgta cgataaatgt agcaaaagcc acatttccat cattaaatgt 29040 aatcccattt ggtgatacag caacatcagc ctgtcatttg ggtcctctga ttgaggggtg 29100 aggatttctg tttgatacct tgtgcataat ggctgcgttc aagcatttaa actcattttt 29160 atttctaacc tacagctgtc atctttgtaa taggatattc atcagaatct tgccagagac 29220 tgtgcatttg ggatcttggg ggatacagca ccaccaccac cctccccctg tccaagagaa 29280 acagatcaac atcttaggtt gagagtctgg ggtctggaag acccgagttc ctgagtgccc 29340 tttgacaagt aacttaaccc ctgtctgcct cagtctcttc atctgtaaag tggggataat 29400 gacagcacct gcttcacagg gttgatggga atccagatgt ggtgggatat agaaaatgct 29460 tattacttcc acctttgaca ccaaatacat ataactaaga gttaactttg gagcagggga 29520 ggaagtgtga ggctccaggc tggaggcaga cctgtgttcg gctgcaagct ggagaggatg 29580 gaccccaaaa gcttggctga tttgaagtcc atccataaaa tggaactcca gagagtttac 29640 acgtttcagt aatgctgcat aacttaatta taagatcttc tctctttgtc ttctttcagt 29700 gttataaaag ctcttttgtc cttgagcttc ctttaccaag aaacatgcat ttatgtatct 29760 ttttgttcat ggaattgccc aagcttgtta gcagatcctt tgtaagaccc aaaagagaca 29820 gacaggggag gagtcttcag atacatataa tcatttttcc caatttccat gttaccagcc 29880 ttgccaggac tttttctcag ttccctgtta cacaatgaaa atagtgtctc tttattgata 29940 attttagtag catcctaatg tggtataaat cgtcttccag agaagaaaat gtgtcagggt 30000 tgcgttatca ctgaggctag ctgggaaagt agatcagccc attagtctga taattcgaag 30060 cgttgtttct gttatttctg aacatcatgt gaactccttt tctgggtgta ttaaaggttt 30120 tcccagtgtg tgtcagtgag actcctgatt gaatttaata tgaataaaga taaattcttt 30180 acatttaagg attaaagtct cagcttctgc ttaacttgag attgcactga gaaactcctg 30240 gctctcgggt atagcggagt cacgacctgg ggatgtctgt cccatatggc tctgtgtgta 30300 agaagaaaaa gctgctgtgg acggagactc tgttcacatt aaatgacatc acctaagcca 30360 tcatgacagc aagaattatt taggaattgc tcagaataaa actgccttca ttatttcata 30420 aaatgtatct tggtatcttt agcaccttat ttatggcttt ttaaaggttc actgggattt 30480 ataaataatt ggacaatgct agagacctag tacaagaatg aaagaggaca ggcttctttc 30540 ttaataacct ttaaacattc atcaggaaga taaaacttta aagcaaaata aaacacatga 30600 aaatagccaa gatgcacaga ccagacaagc aaatactact ttaacttatt tgtatagttc 30660 ttaagagtca catttgttcc tgaagtttca aaatctcggg ctgagtgttt gatcacttag 30720 ggaagtgttg tggccttcac atactcttgt ctcactttga agtctagaaa cacaggtctt 30780 agagcaattt ttatcactgt gagaaagctg aaacttagtg tgagtagctt agtacaattc 30840 agttggccat caaatgtcag aaacaaaact cagtccaggg ccgctggacc cttaggccgg 30900 cgttgttagt ttacaacagt gcctcctggg tccaaacatc taagtgcaca tgtagcaata 30960 gtaaagatag tatgtatgca tacataacac atatgtagag acagcagagt atacgtacac 31020 acatgttgca tacatagcaa cagcagagaa gctcatgaac tataaaggat ggactgtatg 31080 cttgtatcag acattttggt actgacgctt tgtcatatat tgtgtaacat ataaccagct 31140 tgcaatcatc tgcccccaaa gttgaactaa gaaaatccta cagggtacta ggaaaggaag 31200 gccattggga aaaggtggtt atagtggcaa tttgttagct cttatgaatt ttctttttct 31260 ttttagacat actcttaatt ccattttttc aataaatcta tactattttg tgtttttatg 31320 ttagcaagta ctttaagccc ctcaatagaa agttgctaca tcatatagtg attaaaaata 31380 aaaatctctc aaacatacaa gtagaggtgg tatgagactt caaattccct tagccaagta 31440 caagtgcagc agttttgttg gctggctggc tgcatagaag gactgatgga ttggcagacc 31500 ctcaagctgg agtgtaattg atctcattac agaggagcca ggctgggtga cagttgtgct 31560 ttgcaagtgg ttttttgcat tggtgaagta gcccattttg ttgttcctga tgttaaacag 31620 gggatgaagg tattctttta ttggcacaaa cgcgggaaat tgctctggat tcttagagga 31680 tagaacatgt cccctggacg gaataaggtt catgtgtagg gcaaatttag ataggggcac 31740 cttattgggg ttactactgg tctctagatg gtcaaagcaa acaacatgtc catctaagct 31800 gtgatgtcca tctaagctgt gtgtgtccat gagagtgacg cattttctcc tctgcagtgt 31860 tgttatattc taaactgtca gcagacatta attcggtcgc tggtgaagtc ccaccgccta 31920 gagatgaact ctgcctccga tggatgtttt ccacttcagt gccactcgtc tcgcaattac 31980 tgggtcatta atatcattgc atgcaattag tgacagtaga aagagctaga gggttgtggg 32040 atgtgcaccc tccccaccat gaacttttta ctctgaccct ttcccagcta gaccttttcg 32100 tatcttggca aggatatttt aatgattgag actgtcagaa tcttcagagc aggcactgga 32160 ttatgtgctg gaaataattc actcaaacac ctgcttctcc atggttcaga atattttcat 32220 tagatattat cactatccct tccctgggaa gtttcatttt taaaaatctg atgcttaagt 32280 acagctaata tagacaatag ggaattatgt tttatcttta gaactcttac attattcttt 32340 tctttaaaaa tgtgagctga gtcattgcta ttgcagtggt catctggccg cctattttta 32400 aaacacaatt cctctatctt agtagatttt ggcccatatt aagcatatca agaatgactt 32460 tttttttttc aagacatggg gttttattgg gggcttatat acaaggaaag agagagtcca 32520 gtggcagtgg gctggacaag atatccacat ggccctgtgg cagtgagctg ggcaggaaaa 32580 ctgcaactgc ttgcaaacag catgtagttc atctatagca ttttcactta acaccaccca 32640 gctaatgact tccacctggc aaccttcatt taatccagaa cttaggacct cgagtccctg 32700 tacggcccat gttccacagg atgggccgag ggctcagctg ttcctcatag acaaggaatg 32760 actctccaca ttggccactc ccggattccc tagctcagga cacatattca ggtgtgtcta 32820 aggctggctc ttctatgtga agttacttat tcttttacca ttgactctca tgttcccact 32880 atattaagtt tttctgaatt actgtggcaa taagaaacgg tcccttaaat tatactagaa 32940 gaaaagcttt ttttttgttt tgttttttat tttgaaatta tgttaaattt tttttcttaa 33000 ctgagagatt ccacctgcat aaatcgtcat aacttttaac agtaagatct tagacttaga 33060 aagtgatgtt tttcctcaac agaatttatt aaaaatcaag acaccaagct gttccaaaca 33120 atagtttgag gggaaataaa ataaacaact ccataaataa tcttatgttg ttaaacatgt 33180 ctctagcaaa acaaacaaac aaaaaagtcg ggggttgggg gaggtgcagt ttattgccag 33240 tactgtctgg tctttctcag aaaagcgtca gtgtacatca ctgagcctgg acggtatgtt 33300 ttcttgatct atacccccta tgtgtacatg tgcttgcacg cacacacatg tagacacgca 33360 cacatgtgca cctgccatca ctttctgctc ttccgtcttt tcactcttga gtgtctgtag 33420 ccagtagctt tccaggtctg tatagtcaaa gatacctatg gccctgaatg tcttcactga 33480 ttgctatttg acattcatac ggtttttaat ggttaaaagg ctttatgcga aagctgtgat 33540 agaatttctc ctgttctaga tgtggtgttt attgctttat tttgtgactt ttctctcagt 33600 agattgacct tctccctcag tgtccaagcc tcgcatagca tgatggcacc tgtaaactca 33660 gttctgtatc ctggtatcct ttctcttccc aagtagaagc aattaagtaa tatatgtcat 33720 caaaaccttt taagtgcaca tacaaacaaa atcaacttac caaactgctt caaagttgtt 33780 ccatgtttaa cactcttctt tctgagctct gggtagaatg tcctattatt gttcatcatg 33840 aatatttgaa attaaagaaa taaaactgta ccattttctt taagagcatc catttgtact 33900 tgataacatc ttcagtcata tttcaatgct ggcaaagagg aggggagttc taaactgtga 33960 ctcaatttta gaatctactt tttccaaatt attctgttta gtgcagaaaa ctaattaata 34020 gtgttgcata gaaaagtcac tgaagctaag ccagttatta cttcttaatg catgatttac 34080 tgctttaagt tttcaaaaca caaccatagc aatgtggtat taattcaagt gattcttcct 34140 atcatattga acgatatttt cacgggtgaa aaactcacac atcctacatc actgatagtt 34200 tatacagtgt tttagctgtg gctccctgca tgcaaaataa gagttaatca aatgtcagtg 34260 agaaccatct catcaagtag agggcttgtt ttgtttaaat taactttgct aagtataaat 34320 ttcttcttga aaataaattc tgggccgggc gcggtggctc acgcctgtaa tcctagcact 34380 ttgggaggcc gaggcgggcg gatcacgagg tcaggagatc gagaccaaac tggctaacac 34440 tgtgaaaccc cgtctctact aaaaatacaa aaaatgagcc gggtgtggtg gcgggctcct 34500 gtagtcccag ctactcggga ggctgaggca ggagaatggc gtgaacctgg gaggcagagc 34560 ttgtggtgag ccaagatcac accactgcac tccagcctgg gtgacagagc gagactccgt 34620 ctcaaaaaaa aaaaaaagga aaataaattc ttctgtattt ttctttcttc aagtgaggcc 34680 atttagggga aagtatacca taaaacttgc tctaagataa ggcaaatttg gtattatagg 34740 atgaagtgct atgtgatttg aagtaatgct gaatttttta aatatattaa actaaacaag 34800 aataatgagg ccctcggaaa gtcatgatta tatttctcat ttttctcatt ttaaagccac 34860 agtgaaaaac acataaaagg aagaagttag aaaaaaaaat gaatgaaatt ctttttttcc 34920 ttttggcaaa ttaaatagat gtttctgttt cagaagattt tattaattaa ctttaaagaa 34980 acagtcattt atttttggca ttcagtgaac actatcattt ccatgtttag aacttttctt 35040 ctaagttagc atcttaaaag ataactgtga aactcaaggc attcaactac attaatttga 35100 gtttcagaaa ttgaattctt gtttctagag tacatagttt gaattgatgt cagggtgtta 35160 aatagataaa tcttagcttc ctaggttgta tattcacact aattattttt ttatcagcct 35220 tcttattttt caacttacct tattcttttt gtttttttga cactcagatt tgatagccct 35280 gtggtagaag aaaacagtaa tacagtttgg tttgttgttg tgtttgtgtt tattttaaag 35340 tcacggcttt gctttccatg ttgttactgg attatgcttt ttttaattct tcagtttgcc 35400 aagataacag tcttccgatc ttcagaagtc tgtatcaagc ttaaggaaac tgatgtgtag 35460 gaagactcgc ctaagaagtc caaattagca aggctagcat gtgaggacat gctggaaaag 35520 aatagttccc atagatattg acagagaatg ttcataaaat gctacttgtt ttgtggttac 35580 atgagagtaa cttgtgtcca gtgcagctgt atgtaagggc aacgttttta ttctgacgac 35640 tctgtggttt tcatgaccct ggatgcttat catgtctctc tgttggactt cttcaacgga 35700 gttgatacaa atacttgctt ccaagtgtcc atctgccctc tcctccatcc tggccccata 35760 caaatacgct acatttttaa ataatttgaa ataccctcaa tagtatttat atttcctggt 35820 gcttcattct ttccataaga actgtgatac cattattctg taggattttt ttgtgcttcc 35880 ccgtttcaca tctctgtgcc agtgagaccc atatatcggt gcaaatccag aagtttgatt 35940 gtccatctga ttagcacact gttagcaatg tggtggacta aacacagcca agatgtgggg 36000 ctggagctta gcctcctggg agcagagcgg tgaacatcag atgaagacat gtgaaaatgg 36060 agtactactt cctcttcctg gggatgggct aaaaagcaca gccagaaata ttcttgccct 36120 tccagtctgc tttacagtta ctcactggtt ctcttttttt tcctactcag ataaccagta 36180 tactcttccc agtgactaag aactgcagat aagtataggt gcaaatagat ggcaaaccgc 36240 agatggcagc tgtgtggttt cagatgtgct gcagaacttt tagacgatgt gaacgcaagg 36300 aacttttttg ctgagcagta atctctaccc actggaaatt aggccctggg gggaacaatg 36360 tagtgacttc tatatactta ctacatgcag ttagacccct gaagcaaaag cttttaaaaa 36420 caggctgtaa aatgcccatg tatctttatt aagcctattt tccaactgga tagagaaatt 36480 ttctggtaat ttttaaattt gtaaagtcta tttttttcct gagccaaggg aaaaaaaata 36540 tctgggccct aaaagcttag ttataacaat gttatttttt ctatctctga atgattaaat 36600 gtgatttcat ttatgtagca atactatgat tgtggctgca ttagatcacg ctgatagaaa 36660 gatacaaaga aaaactaagt ataatgaact aacaatttat tttcactctt tctctaagtt 36720 aaaaattccc agtacattca aatgaacaat gaaaataatt gcagaattgt ctcctgaaat 36780 ggaaatagat tttttttccc aagcattagc aatttcttgt tatttttcaa aatcagccac 36840 taagcctttc agagcttctt ggtgactatt gcaggagaaa tcagaatatt aatcttgtgg 36900 ttttatttca gagttcgctg ccaggaagga ggtataattg ggataggaga cttttttttt 36960 ttagctgtgt cactgttcaa ggaggggggt ttggaacctc agcataagaa ttacactctg 37020 tgatgaggat gtagcagggg agaagaaagg tgattttcac tatgggaagc tatacttaca 37080 tcaagtataa aatagactga agtcattttg aattacgtta tacttgtaaa gtttacctcc 37140 tggagtttca gttagtacca gtgtactaac tgggttaaaa cagttcatgg caccttagat 37200 catttctaac tcatggcaaa aatctttcct ggtggaacgt gtaactgtat tttaaatgcc 37260 cctttataag caaccaagta tttgggatgt tattttgata ttagtagtga atttttcagt 37320 atcttccagt accctttgca agtcacaggt tgacttaaaa ggaaaagaag caaaatgctg 37380 aatatagcag aaaaactgtc tgcattcaga ctgttcagcc cacttttgct ccccacgtgg 37440 caagcacact cccccaaaca agcaatagcc tgtggcttca gaggaaccta caaaggcagc 37500 atctgtagat ttttccttct tcaactctaa gacttgaatg tttccctctt ccccacacac 37560 ttttttttta aaccaagaaa taaaaaagtt ttcactctta aaggtgcaaa gcagtttcat 37620 tcttatgcaa cacagccttc ctcctactgt cttatagtct gtggatgtta aattatagat 37680 tccaattgaa ttttaatact ctagagattt tacatttgtg gttgtcaaga ccccgttttg 37740 gtaaacctag ggagctccgc acaaaagcat tgatattcag aaaaggcact gacctacaaa 37800 ttaaaagaaa aaaaaatcaa ataatgtgca cctcttgtgc ttccagtttg acaaagcaga 37860 agtcatcagc agtttctccc tctgcagacg cagttctcaa ttctatttac aagtaactgc 37920 tctactgtgc ctgtttttct cttgctgata ctcatttaat tgtttttctt ttggatctga 37980 atctttgact gtcttttccc cctcaagatt aaaataaata catctgtatt cctccccttt 38040 ctttctgtgc actgcccttc agatctcatt ttgtcatttt tcagcttagt gttgaaactt 38100 ttagcaacaa aaagtcagtt acttactttg agtaagtaac tcaaagtaag ttaactttga 38160 gtttgagtgc acttttgcgt gtaggttcat ttatgtgctt gtgaatttaa aaacattggg 38220 attccacctg aatgaagtaa accaaacatt ttaaactatc agccagatag agacatcagc 38280 ctttcacttc tttctatatg cagacatatc ctaatttttt agaaaaatca aataggaaaa 38340 ttctcaacaa ttaattgaag attatagctc tgctctgaaa tggtccagaa ataggatctg 38400 ctcatagaaa ctcatagttt gaagcctctg ggaggaaagg atactttaaa atttagtcac 38460 atatttggag gagggaaaag ggaaagagca gaatgaagaa ctgaaaaaaa tcacacaccg 38520 gggcctgtcg tgaggtgggg gactggggga gggatagcat taggagatat acctaatgta 38580 aatgacgagt taacaggcgc agcccaccaa catggcacac gtatacatat gtaacaaacc 38640 tgcacgttgt gcacatgtac cctagaactt aaagtataat aaaaaaaaat tttaatagcc 38700 ccattaaata attaaaaaga ttttttttag attcacagaa gtgtacaaaa tttttaggtt 38760 tttttttttt taagctgtct gctgaatagt ttcttaatgg tctacaatgt ttgtatctac 38820 aaacagatac tgtctgcttc ttactaccct tccaagacaa gtattattat ggcaattatt 38880 gcccagtttc ccgggaaaaa tttatccaca gttacagaag aatgagatgc aattgtgaga 38940 ctgtaaagtt taagcaagca ctcagagaag cacagtgata tgtatgcaca gaagaggcag 39000 tctttgtttt gaggaaaaca gtgaaagtaa agttaattca agaccacaaa gacaagtaaa 39060 taagtgcctt atttttgtag ttaatataat ttcagtggaa tgcatatttc taccataaat 39120 gcatatagaa cttgtttgct gacctactgt ttggaaaaca aacaatccca ttagaagaat 39180 gtctttggga tttattttta ccagaaaatc aatccttttt tcagtccctt gcaaagtaca 39240 gtgttacaag ccaagacttt gataatcagg tagaaaatgg atttaaattg cagaaatgta 39300 tatgaaacac ttttgttcct tgccccttga actttagggg aatgaaaatg tctagcactc 39360 tccaccttct tttctctcct ggaacttgaa ctgtaattca aagcctgttt ctcattaaag 39420 tacctggcag cctatctctt tacagcttga gttacaaagc tattcagaga cctcgctggt 39480 ctaaagagac agaacaagga tgtgtttaaa tagagcatag gctgttgaaa aaaaaaatgc 39540 tgaaaatggt aaaatgattc tgtccttcct tccactcctc actgctgagg tggagaggga 39600 attcagttgg tgaacaccag caagtggctg gtaaaagtcc ccactttctc tccagggctg 39660 ccacaggacc cagaatgagt ggtgggcatg tgtgtgaacc ctctattcag ccagagtttt 39720 cccgcaacag gtagtttggt tgaagaggtt gactaaggtt gacattggca gtaataacac 39780 gtatgttctt ctgatttaca aaacgatgga ggaaaaaggg gagattttga agacctgatt 39840 tctggtatac ttcttaagca tgcataaggc tgaaaaaaga agacaagggt tgtgggaggc 39900 tcctggtcta gtgtttacag aacttggatg cttgacaaac agagcgtcaa gctaattgtt 39960 cttgaagcag gaaatctgca gtggaggaag caggtgtggg gggatgatta ccacgtttgg 40020 aaatggctgc attaactatt ttgctcttct gagtttggcc ccaaaagagt ccatagactt 40080 tttgaaggat gccatccctt ttatttatag actaacatta aatcagtcat ttgtgaagga 40140 aggagaaagt gcctaaataa atttggagtc agatagcata cgtgcggcag tgtttccgat 40200 atccatttct ctttatttct ttttcttttt ctttttggct ttcagcatcc ccatactttc 40260 agaaaacttg tgactaagag tgaattctta tttttcaaat tgttttcaga catttcatgt 40320 tcatgtaaac ttggcttatt gatttcctga tttttcttta tttttttgtt ttgtccattt 40380 tatttttaat cagctacatc aaatgggtct ttggagggcc tggataacca ggagggaggg 40440 gtgtgccaga caagagccat gaagatcctc atgaaagttg gacaaggtaa agaccatctg 40500 ctgcttcatg acgccactgt gacctggtgt agcccccagc tagtatggtg ctaatgttgc 40560 cgatgcccac cttcattcgc tcttcttttt agttttcaaa gcaaaccctt ctgcactttg 40620 agccactgac agatttcctc aagtcaatgt actaagcttt tattggagat ctaagagtta 40680 agatcagcaa ggtagaatgt ctattgccat agatagatag atagatagat agataataga 40740 tagatagata gatagataga tatttctttt taaaaagcaa aacactttgg ttcaaaatca 40800 aaatatccag aatgaaaact aaaagcttgt gcagttttgc tcatttctga atcttgacta 40860 cagaagagtt ttgttcattg tgacttttcc aatatagata acctattgtg cagaaagaaa 40920 taattattct tctaattaaa aattggtata gtagtcaatc aacttgctca gttaaattga 40980 aatgtcatct gcaatgcttt gcctgccaaa tgcaagaatc cctatagttt ccacagatgg 41040 cctcacgttc taaacctctg aaataactag tataaccatt ttgttttaaa agaaaaatta 41100 tattcttgta tttcacagta ctttgcataa agactcttat gttcattgct attcatgcct 41160 gttgaaatat atatgcagct cctaaagcta gatattgtca gatgtctgtg ccgtaattaa 41220 tcatttgttt ttcatataga tgcaagttct gctggatcaa ccaggaataa agatccaaca 41280 agacgtccag aactagaagc tggtacaaat ggaagaagtt cgacaacaag tccctttgta 41340 aaaccaaatc caggtataac agcatgatct gtgtgtatgg aggtctgtgg gtaccacatt 41400 cttagtagta tcttaaaagg tagggcagag tctaaagact tctaaccagt taggattagc 41460 tggaagttac agtgatcagg aatctttgct gtcagtgagt cattattaat tacactcaat 41520 aagaacaaaa taactcattc caatgaaagt catatattca aaggagtaga gttcatgagc 41580 tgtaagtgcc agttattaga actactctgt caggccaaag gtttcattgg ctgacatttt 41640 atcaagctgg ttgtcaactc cagcttaaag ctgatgttaa tgtatatgta attaatgtgc 41700 taatccctca tctaattata tctaagccac agagggttta attgatcctc ttctaaattt 41760 taaatggtaa catttttaaa tattgcataa tagtattttt tcaggtggtt atcgttattt 41820 tgtttcacat tttccatgta aaagaaaata ttaaacaggt ccctgacaaa agtgtagaat 41880 accagataaa attgtccgtc gttgaccttc gttttcttaa cagtcttgga acaaatagtt 41940 ctgtatttgt taccatgcta atgaaggttt tatagagtag ctgttgagca gacatcagca 42000 gttttgtatt aggattgttg tgtgcttgct tggtcgttgt gcaaatttat cgtctgcagc 42060 aatattccat ccctttccaa gagtcaagga gggaagttgt tatttctaac tttcaatgac 42120 aagatgtgtc aaattcttgt gacaaactga taaatggata atataatgat gccaggcagt 42180 tttttagtgc ttaacatttg ggctggcagt ctgttcggtg tgagagtttc tgctgccttc 42240 caaatatatt ttaagtgtaa atcaaataat acagacgagt tacgagctga acattttccc 42300 aggccccctc actccttccg cgttcccgag ctgttctgtt ctgccaggag gcagggctct 42360 tctttagaag gcaggccctt tgaaggtttg catgaaactc cctttctcaa aggaggcgga 42420 agagcaatac cacataaacg ctcaccgctg acctggagaa ttggccactt ccctttttct 42480 tccctgccgc tgccccaggc tggctgacac gggttagaag atgaagcaag atcaagggct 42540 ggctgtcacc gacagtctgt gctcttgctg gataatgata caaaggaaac cctgtggctt 42600 gggagggtag ggaagtccct cctagagata cctctcattt ccttttgcgt tgagctctta 42660 gacgaggtat tggcgaggca aagtccagct tctagttagt aataagcctg gcttattttt 42720 cacattttta agggtcataa aagcagtccg tctgcactgg gacagcagta actatctctg 42780 accttttctg tctccgcgtc tgcaggttct agcacagacg gcaacagcgc cggacattcg 42840 gggaacaaca tcctcggttc cgaagtggcc ttatttgcag ggattgcttc aggatgcatc 42900 atcttcatcg tcatcatcat cacgctggtg gtcctcttgc tgaagtaccg gaggagacac 42960 aggaagcact cgccgcagca cacgaccacg ctgtcgctca gcacactggc cacacccaag 43020 cgcagcggca acaacaacgg ctcagagccc agtgacatta tcatcccgct aaggactgcg 43080 gacagcgtct tctgccctca ctacgagaag gtcagcggcg actacgggca cccggtgtac 43140 atcgtccagg agatgccccc gcagagcccg gcgaacattt actacaaggt ctgagaggga 43200 ccctggtggt acctgtgctt tcccagagga cacctaatgt cccgatgcct cccttgaggg 43260 tttgagagcc cgcgtgctgg agaattgact gaagcacagc accgggggag agggacactc 43320 ctcctcggaa gagcccgtcg cgctggacag cttacctagt cttgtagcat tcggccttgg 43380 tgaacacaca cgctccctgg aagctggaag actgtgcaga agacgcccat tcggactgct 43440 gtgccgcgtc ccacgtctcc tcctcgaagc catgtgctgc ggtcactcag gcctctgcag 43500 aagccaaggg aagacagtgg tttgtggacg agagggctgt gagcatcctg gcaggtgccc 43560 caggatgcca cgcctggaag ggccggcttc tgcctggggt gcatttcccc cgcagtgcat 43620 accggacttg tcacacggac ctcgggctag ttaaggtgtg caaagatctc tagagtttag 43680 tccttactgt ctcactcgtt ctgttaccca gggctctgca gcacctcacc tgagacctcc 43740 actccacatc tgcatcactc atggaacact catgtctgga gtcccctcct ccagccgctg 43800 gcaacaacag cttcagtcca tgggtaatcc gttcatagaa attgtgtttg ctaacaaggt 43860 gccctttagc cagatgctag gctgtctgcg aagaaggcta ggagttcata gaagggagtg 43920 gggctgggga aagggctggc tgcaattgca gctcactgct gctgcctctg aaacagaaag 43980 ttggaaagga aaaaagaaaa aagcaattag gtagcacagc actttggttt tgctgagatc 44040 gaagaggcca gtaggagaca cgacagcaca cacagtggat tccagtgcat ggggaggcac 44100 tcgctgttat caaatagcga tgtgcaggaa gaaaagcccc tcttcattcc ggggaacaaa 44160 gacgggtatt gttgggaaag gaacaggctt ggagggaagg gagaaagtag gccgctgatg 44220 atatattcgg gcaggactgt tgtggtactg gcaataagat acacagctcc gagctgtagg 44280 agagtcggtc tgctttggat gattttttaa gcagactcag ctgctatact tatcacattt 44340 tattaaacac agggaaagca tttaggagaa tagcagagag ccaaatctga cctaaaagtt 44400 gaaaagccaa aggtcaaaca ggctgtaatt ccatcatcat cgttgttatt aaagaatcct 44460 tatctataaa aggtaggtca gatccccctc cccccaggtt cctccttccc ctcccgattg 44520 agccttacga cactttggtt tatgcggtgc tgtccgggtg ccagggctgc agggtcggta 44580 ctgatggagg ctgcagcgcc cggtgctctg tgtcaaggtg aagcacatac ggcagacctc 44640 ttagagtcct taagacggaa gtaaattatg atgtccaggg ggagaaggaa gataggacgt 44700 atttataata ggtatataga acacaaggga tataaaatga aagattttta ctaatatata 44760 ttttaaggtt gcacacagta cacaccagaa gatgtgaaat tcatttgtgg caattaagtg 44820 gtcccaatgc tcagcgctta aaaaaacaaa ttggacagct acttctggga aaaacaacat 44880 cattccaaaa agaacaataa tgagagcaaa tgcaaaaata accaagtcct ccgaaggcat 44940 ctcacggaac cgtagactag gaagtacgag ccccacagag caggaagccg atgtgactgc 45000 atcatatatt taacaatgac aagatgttcc ggcgtttatt tctgcgttgg gttttccctt 45060 gccttatggg ctgaagtgtt ctctagaatc cagcaggtca cactgggggc ttcaggtgac 45120 gatttagctg tggctccctc ctcctgtcct cccccgcacc ccctcccttc tgggaaacaa 45180 gaagagtaaa caggaaacct actttttatg tgctatgcaa aatagacatc tttaacatag 45240 tcctgttact atggtaacac tttgctttct gaattggaag ggaaaaaaaa tgtagcgaca 45300 gcattttaag gttctcagac ctccagtgag tacctgcaaa aatgagttgt cacagaaatt 45360 atgatcctct atttcctgaa cctggaaatg atgttggtcc aaagtgcgtg tgtgtatgtg 45420 tgagtgggtg cgtggtatac atgtgtacat atatgtataa tatatatcta caatatatat 45480 tatatatatc tatatcatat ttctgtggag ggttgccatg gtaaccagcc acagtacata 45540 tgtaattctt tccatcaccc caacctctcc tttctgtgca ttcatgcaag agtttcttgt 45600 aagccatcag aagttacttt taggatgggg gagaggggcg agaaggggaa aaatgggaaa 45660 tagtctgatt ttaatgaaat caaatgtatg tatcatcagt tggctacgtt ttggttctat 45720 gctaaactgt gaaaaatcag atgaattgat aaaagagttc cctgcaacca attgaaaagt 45780 gttctgtgcg tctgttttgt gtctggtgca gaatatgaca atctaccaac tgtccctttg 45840 tttgaagttg gtttagcttt ggaaagttac tgtaaatgcc ttgcttgtat gatcgtccct 45900 ggtcacccga ctttggaatt tgcaccatca tgtttcagtg aagatgctgt aaataggttc 45960 agattttact gtctatggat ttggggtgtt acagtagcct tattcacctt tttaataaaa 46020 atacacatga aaacaagaaa gaaatggctt ttcttaccca gattgtgtac atagagcaat 46080 gttggttttt tataaagtct aagcaagatg ttttgtataa aatctgaatt ttgcaatgta 46140 tttagctaca gcttgtttaa cggcagtgtc attccccttt gcactgtaat gaggaaaaaa 46200 tggtataaaa ggttgccaaa ttgctgcata tttgtgccgt aattatgtac catgaatatt 46260 tatttaaaat ttcgttgtcc aatttgtaag taacacagta ttatgcctga gttataaata 46320 tttttttctt tctttgtttt attttaatag cctgtcatag gttttaaatc tgctttagtt 46380 tcacattgca gttagcccca gaaaatgaaa tccgtgaagt cacattccac atctgtttca 46440 aactgaattt gttcttaaaa aaataaaata tttttttcct atggaaaaag tgccttcaaa 46500 gtacttttct tcttttcttt tcatttttct ttcttttttg tttgttattt taatttgcct 46560 tcctttactc tatttccccc aaaattacag ttaatccaga gacttctgtc tatggacact 46620 gtgtgcccac ttttcaaatc gggatgtgca catcacacaa atattaattt tgctaagagc 46680 cgtgagctcg ttattcccca taaaaccaca aactctcatt tcaaaaataa tatgcaattg 46740 tataaaatcc taataatcct caacagaaaa tctctctagt ggcacctgaa ttgcacagtc 46800 agaatagtta cctacaaagt tagctgtctc aggaaaaaga gggaaatgta ctcttatctt 46860 acacttaatt ccagatcgat ctagtgtcca tagaagcaaa gcagcggtct atcgcttcat 46920 ttttcttaac agaataatcc tagggagaag gaaaaagtca aatgtttttg taaaaactta 46980 acattgatga gagcacattt tgagatactg gttgaaggca agccagaggc cagctgtgag 47040 tttaaaacac actttcagtg gtcatcttcc tggggaaggt aactatgtac cctgaggggt 47100 gtgattgttt ttcaggcctc ttgtagtcct gggtattaag actgatcact atacttcctg 47160 ggtagctttt taaacaacca agagtcccca cccatggcag agaacttcac acccttgaga 47220 tctgagtgct aacatcttta cattatctca ggttaatccc tgccataaga gtgagacatt 47280 ttaggtaggg agaataggaa aaggcccctg gagtcaggaa gcaagccaga agagttccca 47340 ggtctgactg acttgcccag gaccacaccc cagtcagtgg aagaaaccat atgtttcaaa 47400 agtccatagg tgatgaagaa aggctggttt ggacacaggg atctacacag cttagcacag 47460 tacttgtact agagaaatgt ttgttaaata aatgcctccc ttcatcagca acaacaacaa 47520 tccccggagg cccagggttt tcgtggatgg tagaatcgta gtttcatctg ttccaggatt 47580 ttctcctgct gcacccacct gccttcagtt gcagatccaa gttgatggat gcaattttat 47640 ggccaagcca ctaagctgaa ccctcgaccg tgtgcaccaa ggtcatttct agttagtttg 47700 ggcaaaagaa aagaaccaaa gtgaaatcat gcattttcaa cctttacttt gtggtaccta 47760 ttacaacata aattaccgaa tacacaatgt catggtttgg accctgtttt taaaccacgt 47820 tttctgagta agccaagctc agctaagaaa caagatttgt ccacttgttc ttttcacctg 47880 ggaagaagga acacacgcag aagctccttg cagagggcgt ataaccagat gccctttgtt 47940 ctgtctcaaa ggtgagccca gcctgctctg agcacgttac ttctctgcac aggcgagcgt 48000 ccctcagcca gcagccagct ctctttctta tcaactccct ttttggcaaa gcagatggcc 48060 attcaaaccc tgggataaag gttcgcaatg gaaatgagaa aaaataaagt attcctttct 48120 gggtaactgc tgtccagctt actgtcagtc tctgaaccca acccagagaa gaattagcaa 48180 gagaaaaagt aaactccaga gtagtaaaac tcactttcct aatgtaggtt aaaaaaaaaa 48240 aagaaagaaa aagaaaaagg accattaccc tactttttgg ttgggtagca aataggtctc 48300 aggtggctca ggagacacag tagctactgc agcaccaagg acactagcct ggatgtgcta 48360 ctgctttggt ttttactttg gatggatttt tgaattacgt ttgtgatatt tacactatac 48420 acttacttga aggcaaattt tcagtgttgt catggaaaag agtatttcct actccgattc 48480 attattccag gtagagaatc attcatcagt gatttgaggg gggggtacta aaatattgca 48540 ttatttgtaa caaaatatag caaaccaggt aggttaatcc atttttagat atgtcccaaa 48600 agctaacgat acatgcagct taagattttt atactttgat aacttgatta gtggaatctc 48660 tttaaaacag tgttcatatg aagtcaacca tatctttaaa aacaaccaac cccctcaaga 48720 attctccagg tatacaccag gtacacaggc atacggcaca aggaaaataa tactaattat 48780 agcagcacag tttctggcca gttgtagaca gttaacaaag gtgttaaatc atgaaagggg 48840 cacctggttt ctggctgtat ttctgctact gactcatgct gtgaccttca gcagatcctt 48900 ttatgttctg tgcctcagag cctcacctgt aaagtgagag ggatggattc taagatacca 48960 aaggtcgctt ttgccttttt gcaggattcg cagcaaaaag c 49001 12 413 DNA H. sapiens unsure 12 unknown 12 gcgcagcgcc tncggagctg cctgcgggcg cacgccgtct tccccgccag tntgccccgg 60 aggattgggg gtcccagcct gcgtcccgtc agtcccttct tggcccggag tgcgcggant 120 gggagtggct tcgccatggc tgtaagaagg gactccgtgt ggaagtactg ctggggtgtt 180 ttnatggttt tatgcagaac tgcgatttcc aaatcgatag ttttagagcc tatctattgg 240 aattcctcga actccaaatt tctacctggg acaaggactg gtactatacc cacagatagg 300 agacaaattg gatattattt gccccaaagt ggactctaaa actgttggcc agtatgaata 360 ttataaagtt tatattggtt gataaagacc aagcagacag atgcactatt aag 413 13 20 DNA Artificial Sequence Antisense Oligonucleotide 13 aaccactgtc ttcccttggc 20 14 20 DNA Artificial Sequence Antisense Oligonucleotide 14 aagtgtcgta aggctcaatc 20 15 20 DNA Artificial Sequence Antisense Oligonucleotide 15 ttaataacaa cgatgatgat 20 16 20 DNA Artificial Sequence Antisense Oligonucleotide 16 tcatccaaag cagaccgact 20 17 20 DNA Artificial Sequence Antisense Oligonucleotide 17 caacagtttt agagtccact 20 18 20 DNA Artificial Sequence Antisense Oligonucleotide 18 ccagtaccac aacagtcctg 20 19 20 DNA Artificial Sequence Antisense Oligonucleotide 19 cagtcctgcc cgaatatatc 20 20 20 DNA Artificial Sequence Antisense Oligonucleotide 20 ccactgtgtg tgctgtcgtg 20 21 20 DNA Artificial Sequence Antisense Oligonucleotide 21 gtcgtaaggc tcaatcggga 20 22 20 DNA Artificial Sequence Antisense Oligonucleotide 22 atgatgcagt cacatcggct 20 23 20 DNA Artificial Sequence Antisense Oligonucleotide 23 tgatgacgat gaagatgatg 20 24 20 DNA Artificial Sequence Antisense Oligonucleotide 24 cctgccagga tgctcacagc 20 25 20 DNA Artificial Sequence Antisense Oligonucleotide 25 taaagggcac cttgttagca 20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26 tccttgtcca ggtagaaatt 20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27 gaggacttgg ttatttttgc 20 28 20 DNA Artificial Sequence Antisense Oligonucleotide 28 ggatcttcat ggctcttgtc 20 29 20 DNA Artificial Sequence Antisense Oligonucleotide 29 gctgcaggct ccatcagtac 20 30 20 DNA Artificial Sequence Antisense Oligonucleotide 30 ccagacatga gtgttccatg 20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31 ggcatcggga cattaggtgt 20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32 tttccctgtg tttaataaaa 20 33 20 DNA Artificial Sequence Antisense Oligonucleotide 33 ctttctgttt cagaggcagc 20 34 20 DNA Artificial Sequence Antisense Oligonucleotide 34 cagaacttgc atcttgtcca 20 35 20 DNA Artificial Sequence Antisense Oligonucleotide 35 tctagagaac acttcagccc 20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36 ctattataaa tacgtcctat 20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37 caaaccctca agggaggcat 20 38 20 DNA Artificial Sequence Antisense Oligonucleotide 38 cacggagtcc cttctcacag 20 39 20 DNA Artificial Sequence Antisense Oligonucleotide 39 cagggtccct ctcagacctt 20 40 20 DNA Artificial Sequence Antisense Oligonucleotide 40 ttaggtgtcc tctgggaaag 20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41 acagccctct cgtccacaaa 20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42 actgtcttcc cttggcttct 20 43 20 DNA Artificial Sequence Antisense Oligonucleotide 43 tctagacccc agaggttagg 20 44 20 DNA Artificial Sequence Antisense Oligonucleotide 44 tcttggtctg gtttggcaca 20 45 20 DNA Artificial Sequence Antisense Oligonucleotide 45 agaaatttgg agttcgagga 20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46 agaccgactc tcctacagct 20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47 agcaatccct gcaaataagg 20 48 20 DNA Artificial Sequence Antisense Oligonucleotide 48 tctgcacagt cttccagctt 20 49 20 DNA Artificial Sequence Antisense Oligonucleotide 49 aggtagaaat ttggagttcg 20 50 20 DNA Artificial Sequence Antisense Oligonucleotide 50 ttctcacagc catggctgtg 20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51 tttggaaatc gcagttctgc 20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52 tgggtatagt accagtcctt 20 53 20 DNA Artificial Sequence Antisense Oligonucleotide 53 tgatggtgaa tttgatatct 20 54 20 DNA Artificial Sequence Antisense Oligonucleotide 54 catttgatgt agatataatg 20 55 20 DNA Artificial Sequence Antisense Oligonucleotide 55 tgctagaacc tggatttggt 20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56 gagtgagaca gtaaggacta 20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57 tgaggtgctg cagagccctg 20 58 20 DNA Artificial Sequence Antisense Oligonucleotide 58 gaacggatta cccatggact 20 59 20 DNA Artificial Sequence Antisense Oligonucleotide 59 gccctggcac ccggacagca 20 60 20 DNA Artificial Sequence Antisense Oligonucleotide 60 gacatcataa tttacttccg 20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61 cataaggcaa gggaaaaccc 20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62 gatgtgtctt agctatggat 20 63 20 DNA Artificial Sequence Antisense Oligonucleotide 63 cactggactc tctctttcct 20 64 20 DNA Artificial Sequence Antisense Oligonucleotide 64 gctaggatta caggcgtgag 20 65 20 DNA Artificial Sequence Antisense Oligonucleotide 65 tggcacagag atgtgaaacg 20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66 tggtctttac cttgtccaac 20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67 gaacttgcat ctatatgaaa 20 68 20 DNA Artificial Sequence Antisense Oligonucleotide 68 gctgttatac ctggatttgg 20 69 20 DNA Artificial Sequence Antisense Oligonucleotide 69 gtgctagaac ctgcagacgc 20 70 20 DNA Artificial Sequence Antisense Oligonucleotide 70 agcccccagt gtgacctgct 20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71 gagccacagc taaatcgtca 20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72 tttccaggtt caggaaatag 20 73 20 DNA Artificial Sequence Antisense Oligonucleotide 73 attgtagata tatattatac 20 74 20 DNA Artificial Sequence Antisense Oligonucleotide 74 tgtactgtgg ctggttacca 20 75 20 DNA Artificial Sequence Antisense Oligonucleotide 75 tacatatgta ctgtggctgg 20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76 aactcttgca tgaatgcaca 20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77 cccccatcct aaaagtaact 20 78 20 DNA Artificial Sequence Antisense Oligonucleotide 78 taaaatctga acctatttac 20 79 20 DNA Artificial Sequence Antisense Oligonucleotide 79 attgctctat gtacacaatc 20 80 20 DNA Artificial Sequence Antisense Oligonucleotide 80 ggcaaccttt tataccattt 20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81 cacaaatatg cagcaatttg 20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82 agcagattta aaacctatga 20 83 20 DNA Artificial Sequence Antisense Oligonucleotide 83 tgcaatgtga aactaaagca 20 84 20 DNA Artificial Sequence Antisense Oligonucleotide 84 cgcaggctgg gacccccaat 20 85 20 DNA H. sapiens 85 gccaagggaa gacagtggtt 20 86 20 DNA H. sapiens 86 gattgagcct tacgacactt 20 87 20 DNA H. sapiens 87 agtggactct aaaactgttg 20 88 20 DNA H. sapiens 88 caggactgtt gtggtactgg 20 89 20 DNA H. sapiens 89 cacgacagca cacacagtgg 20 90 20 DNA H. sapiens 90 tcccgattga gccttacgac 20 91 20 DNA H. sapiens 91 agccgatgtg actgcatcat 20 92 20 DNA H. sapiens 92 gctgtgagca tcctggcagg 20 93 20 DNA H. sapiens 93 tgctaacaag gtgcccttta 20 94 20 DNA H. sapiens 94 gcaaaaataa ccaagtcctc 20 95 20 DNA H. sapiens 95 gacaagagcc atgaagatcc 20 96 20 DNA H. sapiens 96 gtactgatgg agcctgcagc 20 97 20 DNA H. sapiens 97 catggaacac tcatgtctgg 20 98 20 DNA H. sapiens 98 acacctaatg tcccgatgcc 20 99 20 DNA H. sapiens 99 ttttattaaa cacagggaaa 20 100 20 DNA H. sapiens 100 gctgcctctg aaacagaaag 20 101 20 DNA H. sapiens 101 tggacaagat gcaagttctg 20 102 20 DNA H. sapiens 102 gggctgaagt gttctctaga 20 103 20 DNA H. sapiens 103 atgcctccct tgagggtttg 20 104 20 DNA H. sapiens 104 ctgtgagaag ggactccgtg 20 105 20 DNA H. sapiens 105 ctttcccaga ggacacctaa 20 106 20 DNA H. sapiens 106 agaagccaag ggaagacagt 20 107 20 DNA H. sapiens 107 cctaacctct ggggtctaga 20 108 20 DNA H. sapiens 108 tgtgccaaac cagaccaaga 20 109 20 DNA H. sapiens 109 agctgtagga gagtcggtct 20 110 20 DNA H. sapiens 110 gcagaactgc gatttccaaa 20 111 20 DNA H. sapiens 111 aaggactggt actataccca 20 112 20 DNA H. sapiens 112 agatatcaaa ttcaccatca 20 113 20 DNA H. sapiens 113 accaaatcca ggttctagca 20 114 20 DNA H. sapiens 114 tagtccttac tgtctcactc 20 115 20 DNA H. sapiens 115 cagggctctg cagcacctca 20 116 20 DNA H. sapiens 116 agtccatggg taatccgttc 20 117 20 DNA H. sapiens 117 tgctgtccgg gtgccagggc 20 118 20 DNA H. sapiens 118 cggaagtaaa ttatgatgtc 20 119 20 DNA H. sapiens 119 gggttttccc ttgccttatg 20 120 20 DNA H. sapiens 120 aggaaagaga gagtccagtg 20 121 20 DNA H. sapiens 121 ctcacgcctg taatcctagc 20 122 20 DNA H. sapiens 122 cgtttcacat ctctgtgcca 20 123 20 DNA H. sapiens 123 gcgtctgcag gttctagcac 20 124 20 DNA H. sapiens 124 agcaggtcac actgggggct 20 125 20 DNA H. sapiens 125 tgacgattta gctgtggctc 20 126 20 DNA H. sapiens 126 ctatttcctg aacctggaaa 20 127 20 DNA H. sapiens 127 tggtaaccag ccacagtaca 20 128 20 DNA H. sapiens 128 ccagccacag tacatatgta 20 129 20 DNA H. sapiens 129 tgtgcattca tgcaagagtt 20 130 20 DNA H. sapiens 130 aaatggtata aaaggttgcc 20 131 20 DNA H. sapiens 131 caaattgctg catatttgtg 20 132 20 DNA H. sapiens 132 tcataggttt taaatctgct 20 133 20 DNA H. sapiens 133 tgctttagtt tcacattgca 20 134 20 DNA H. sapiens 134 attgggggtc ccagcctgcg 20 

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targeted to a nucleic acid molecule encoding Ephrin-B2, wherein said compound specifically hybridizes with said nucleic acid molecule encoding Ephrin-B2 (SEQ ID NO: 4) and inhibits the expression of Ephrin-B2.
 2. The compound of claim 1 comprising 12 to 50 nucleobases in length.
 3. The compound of claim 2 comprising 15 to 30 nucleobases in length.
 4. The compound of claim 1 comprising an oligonucleotide.
 5. The compound of claim 4 comprising an antisense oligonucleotide.
 6. The compound of claim 4 comprising a DNA oligonucleotide.
 7. The compound of claim 4 comprising an RNA oligonucleotide.
 8. The compound of claim 4 comprising a chimeric oligonucleotide.
 9. The compound of claim 4 wherein at least a portion of said compound hybridizes with RNA to form an oligonucleotide-RNA duplex.
 10. The compound of claim 1 having at least 70% complementarity with a nucleic acid molecule encoding Ephrin-B2 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of Ephrin-B2.
 11. The compound of claim 1 having at least 80% complementarity with a nucleic acid molecule encoding Ephrin-B2 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of Ephrin-B2.
 12. The compound of claim 1 having at least 90% complementarity with a nucleic acid molecule encoding Ephrin-B2 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of Ephrin-B2.
 13. The compound of claim 1 having at least 95% complementarity with a nucleic acid molecule encoding Ephrin-B2 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of Ephrin-B2.
 14. The compound of claim 1 having at least one modified internucleoside linkage, sugar moiety, or nucleobase.
 15. The compound of claim 1 having at least one 2′-O-methoxyethyl sugar moiety.
 16. The compound of claim 1 having at least one phosphorothioate internucleoside linkage.
 17. The compound of claim 1 having at least one 5-methylcytosine.
 18. A method of inhibiting the expression of Ephrin-B2 in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of Ephrin-B2 is inhibited.
 19. A method of screening for a modulator of Ephrin-B2, the method comprising the steps of: a. contacting a preferred target segment of a nucleic acid molecule encoding Ephrin-B2 with one or more candidate modulators of Ephrin-B2, and b. identifying one or more modulators of Ephrin-B2 expression which modulate the expression of Ephrin-B2.
 20. The method of claim 19 wherein the modulator of Ephrin-B2 expression comprises an oligonucleotide, an antisense oligonucleotide, a DNA oligonucleotide, an RNA oligonucleotide, an RNA oligonucleotide having at least a portion of said RNA oligonucleotide capable of hybridizing with RNA to form an oligonucleotide-RNA duplex, or a chimeric oligonucleotide.
 21. A diagnostic method for identifying a disease state comprising identifying the presence of Ephrin-B2 in a sample using at least one of the primers comprising SEQ ID NOs 5 or 6, or the probe comprising SEQ ID NO:
 7. 22. A kit or assay device comprising the compound of claim
 1. 23. A method of treating an animal having a disease or condition associated with Ephrin-B2 comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of Ephrin-B2 is inhibited.
 24. The method of claim 23 wherein the disease or condition is a hyperproliferative disorder. 